Anti-complement factor C1q antibodies and uses thereof

ABSTRACT

The present invention provides anti-C1q antibodies and methods of using the same.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/989,038, filed Jan. 6, 2016, which is a continuation applicationof PCT/US14/046042, filed Jul. 9, 2014, which claims the benefit of U.S.Provisional Application No. 61/844,369, filed Jul. 9, 2013, and U.S.Provisional Application No. 61/871,813, filed Aug. 29, 2013. The entirecontents of U.S. application Ser. No. 14/989,038, PCT/US14/046042, U.S.Provisional Application No. 61/844,369, and U.S. Provisional ApplicationNo. 61/871,813 are hereby incorporated herein in their entirety by thisreference.

GOVERNMENT GRANTS

This invention was made with government support under Grant NumberR43AG043302, awarded by the National Institute On Aging of the NationalInstitutes of Health. The government has certain rights in theinvention.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 717192000640SeqList.txt,date recorded: Jul. 9, 2014, size: 21 KB).

BACKGROUND 1. Field

The present disclosure relates to anti-C1q antibodies and methods ofusing the same.

2. Description of Related Art

Excessive complement activation has been associated with a range ofdisease conditions, including numerous inflammatory and autoimmunediseases. More recently, the complement system has also been shown tocontribute to neurodegenerative disease pathology. Specifically,complement factors, such as C1q, were shown to be expressed in neuronalsynapses and to mark these synapses for elimination. See, e.g., U.S.Patent Publication Nos. 2012/0195880 and 2012/328601. While selectivesynapse loss is an essential aspect of normal brain development(“synaptic pruning”), excessive synapse loss, especially in a mature oraging brain, results in neurodegeneration and cognitive decline.Elevated synaptic complement expression was found to contribute tosynaptic loss in normal aging and in neurodegenerative diseaseprogression. Conversely, lowering neuronal complement expression wasfound to be neuroprotective. Based on these findings, neutralizing theactivity of complement factors such as C1q is regarded as a promisingtherapeutic strategy to prevent synapse loss and to slowneurodegenerative disease progression as well as cognitive decline innormal aging.

Neurodegenerative diseases involving synapse loss and considered to beamenable to treatments aiming at the neutralization of complementfactors such as C1q include Alzheimer's disease, amyotrophic lateralsclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, Downsyndrome, Parkinson's disease, Huntington's disease, and the like.

Only a limited number of complement neutralizing antibodies are known todate (see, e.g., Klos A. et al., Mol Immunol. 2009, 46(14), 2753-2766;Carroll S. & Georgiou G., Immunobiology 2013, 218(8), 1041-1048; Tuzunet al., J. Neuroimmunol. 2007, 182, 167-176; Nelson et al., J. Clin.Invest. 2006, 116:2892-2900; Heinz et al., J. Immunol. 1984, 133,400-404; Jiang et al., J. Immunol. 1991, 146, 2324-2330; Trinder et al.,Scand. J. Immunol. 1999, 50, 635-641; Hwang et al., Mol. Immunol. 2008,45, 2570-2580). Only the C5 neutralizing antibody Eculizumab, aninhibitor of the terminal complement activation pathway, has obtainedregulatory approval to date; Eculizumab is marketed for the treatment ofparoxysmal nocturnal hemoglobinuria (PNH; Hillmen et al., N Engl J Med.2006, 355(12):1233-43).

Thus, there is a need to develop further antibodies that specificallybind to and neutralize biological activities of complement factors suchas C1q.

All references cited herein, including patent applications andpublications, are hereby incorporated by reference in their entirety.

BRIEF SUMMARY

Provided herein are anti-C1q antibodies and methods of using anti-C1qantibodies.

In certain aspects, this disclosure provides for an isolated anti-C1qantibody comprising a light chain variable domain and a heavy chainvariable domain, wherein the light chain variable domain comprises theHVR-L1, HVR-L2, and HVR-L3 of the monoclonal antibody M1 produced by ahybridoma cell line deposited with Accession Number PTA-120399 orprogeny thereof; and/or wherein the heavy chain variable domaincomprises the HVR-H1, HVR-H2, and HVR-H3 of the monoclonal antibody M1produced by a hybridoma cell line deposited with ATCC Accession NumberPTA-120399 or progeny thereof.

In certain aspects, the present disclosure provides for an isolatedantibody that specifically binds to a C1q protein, wherein the antibodycomprises a light chain HVR selected from the group consisting of HVR-L1comprising the amino acid sequence of SEQ ID NO:5, HVR-L2 comprising theamino acid sequence of SEQ ID NO:6, and HVR-L3 comprising the amino acidsequence of SEQ ID NO:7; and/or wherein the antibody comprises a heavychain HVR selected from the group consisting of HVR-H1 comprising theamino acid sequence of SEQ ID NO:9, HVR-H2 comprising the amino acidsequence of SEQ ID NO:10, and HVR-H3 comprising the amino acid sequenceof SEQ ID NO:11. In some embodiments that may be combined with any ofthe preceding embodiments, the antibody comprises HVR-L1 comprising theamino acid sequence of SEQ ID NO:5, HVR-L2 comprising the amino acidsequence of SEQ ID NO:6, and HVR-L3 comprising the amino acid sequenceof SEQ ID NO:7; and/or wherein the antibody comprises HVR-H1 comprisingthe amino acid sequence of SEQ ID NO:9, HVR-H2 comprising the amino acidsequence of SEQ ID NO:10, and HVR-H3 comprising the amino acid sequenceof SEQ ID NO:11. In some embodiments that may be combined with any ofthe preceding embodiments, the antibody comprises a light chain variabledomain comprising an amino acid sequence that is at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQID NO:4. In some embodiments that may be combined with any of thepreceding embodiments, the antibody comprises a heavy chain variabledomain comprising an amino acid sequence that is at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQID NO:8.

In certain aspects, the present disclosure provides for an isolatedmurine anti-human C1q monoclonal antibody M1 produced by a hybridomacell line deposited with ATCC Accession Number PTA-120399, or progenythereof.

In some embodiments that may be combined with any of the precedingembodiments, the antibody binds specifically to both human C1q and mouseC1q. In some embodiments that may be combined with any of the precedingembodiments, the antibody binds specifically to rat C1q. In someembodiments that may be combined with any of the preceding embodiments,the antibody binds specifically to human C1q, mouse C1q, and rat C1q. Insome embodiments that may be combined with any of the precedingembodiments, the antibody has dissociation constant (K_(D)) for humanC1q and mouse C1q that ranges from less than about 30 nM to less thanabout 100 pM. In some embodiments that may be combined with any of thepreceding embodiments, the antibody has dissociation constant (K_(D))for human C1q and mouse C1q of less than about 30 nM. In someembodiments that may be combined with any of the preceding embodiments,the antibody has dissociation constant (K_(D)) for human C1q and mouseC1q of less than about 20 nM. In some embodiments that may be combinedwith any of the preceding embodiments, the antibody has dissociationconstant (K_(D)) for human C1q and mouse C1q of less than about 10 nM.In some embodiments that may be combined with any of the precedingembodiments, the antibody has dissociation constant (K_(D)) for humanC1q and mouse C1q of less than about 5 nM. In some embodiments that maybe combined with any of the preceding embodiments, the antibody hasdissociation constant (K_(D)) for human C1q and mouse C1q of less thanabout 1 nM. In some embodiments that may be combined with any of thepreceding embodiments, the antibody has dissociation constants (K_(D))for human C1q and mouse C1q of less than 100 pM or less than about 100pM. In some embodiments that may be combined with any of the precedingembodiments, the antibody specifically binds to and neutralizes abiological activity of C1q. In some embodiments that may be combinedwith any of the preceding embodiments, the biological activity of C1q is(1) C1q binding to an autoantibody, (2) C1q binding to C1r, (3) C1qbinding to C1s, (4) C1q binding to phosphatidylserine, (5) C1q bindingto pentraxin-3, (6) C1q binding to C-reactive protein (CRP), (7) C1qbinding to globular C1q receptor (gC1qR), (8) C1q binding to complementreceptor 1 (CR1), (9) C1q binding to B-amyloid, or (10) C1q binding tocalreticulin. In some embodiments that may be combined with any of thepreceding embodiments, the biological activity of C1q is (1) activationof the classical complement activation pathway, (2) activation ofantibody and complement dependent cytotoxicity, (3) CH50 hemolysis, (4)synapse loss, (5) B-cell antibody production, (6) dendritic cellmaturation, (7) T-cell proliferation, (8) cytokine production (9)microglia activation, (10) Arthus reaction, (11) phagocytosis ofsynapses or nerve endings or (12) activation of complement receptor 3(CR3/C3) expressing cells. In some embodiments that may be combined withany of the preceding embodiments, CH50 hemolysis comprises human, mouse,and/or rat CH50 hemolysis. In some embodiments that may be combined withany of the preceding embodiments, the antibody is capable ofneutralizing at least 50%, at least 80%, or at least 90% of CH50hemolysis. In some embodiments that may be combined with any of thepreceding embodiments, the antibody is capable of neutralizing at least50% of CH50 hemolysis at a dose of less than 200 ng/ml, less than 100ng/ml, less than 50 ng/ml, or less than 20 ng/ml. In some embodimentsthat may be combined with any of the preceding embodiments, the antibodyis a murine antibody. In some embodiments that may be combined with anyof the preceding embodiments, the antibody is a humanized or chimericantibody.

In certain aspects, the present disclosure provides for an isolatedanti-C1q antibody which binds essentially the same C1q epitope as theantibody M1 produced by the hybridoma cell line with ATCC AccessionNumber PTA-120399 or anti-C1q binding fragments thereof. In someembodiments, the antibody comprises a light chain variable domaincomprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:5,an HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and anHVR-L3 comprising the amino acid sequence of SEQ ID NO:7. In someembodiments, the light chain variable domain comprises a light chainvariable domain comprising an amino acid sequence that is at least 85%,at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identicalto SEQ ID NO:4. In some embodiments, the antibody comprises a heavychain variable domain comprising an HVR-H1 comprising the amino acidsequence of SEQ ID NO:9, an HVR-H2 comprising the amino acid sequence ofSEQ ID NO:10, and an HVR-H3 comprising the amino acid sequence of SEQ IDNO:11. In some embodiments, the heavy chain variable domain comprises anamino acid sequence that is at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to SEQ ID NO:8.

In certain aspects, the present disclosure provides an isolated anti-C1qantibody, which binds to a C1q protein and binds to one or more aminoacids of the C1q protein within amino acid residues selected from thegroup consisting of: i. amino acid residues 96-226 of SEQ ID NO:1 (SEQID NO:16), or amino acid residues of a C1q protein chain A (C1qA)corresponding to amino acid residues 196-226(GLFQVVSGGMVLQLQQGDQVWVEKDPKKGHI) of SEQ ID NO:1 (SEQ ID NO:16); ii.amino acid residues 196-221 of SEQ ID NO:1 (SEQ ID NO:17), or amino acidresidues of a C1qA corresponding to amino acid residues 196-221(GLFQVVSGGMVLQLQQGDQVWVEKDP) of SEQ ID. NO:1 (SEQ ID NO:17); iii. aminoacid residues 202-221 of SEQ ID NO:1 (SEQ ID NO:18), or amino acidresidues of a C1qA corresponding to amino acid residues 202-221(SGGMVLQLQQGDQVWVEKDP) of SEQ ID NO:1 (SEQ ID NO:18); iv. amino acidresidues 202-219 of SEQ ID NO:1 (SEQ ID NO:19), or amino acid residuesof a C1qA corresponding to amino acid residues 202-219SGGMVLQLQQGDQVWVEK of SEQ ID NO:1 (SEQ ID NO:19); and v. amino acidresidues Lys 219 and/or Ser 202 of SEQ ID NO:1, or amino acid residuesof a C1qA corresponding Lys 219 and/or Ser 202 of SEQ ID NO:1. In someembodiments, the antibody further binds to one or more amino acids ofthe C1q protein within amino acid residues selected from the groupconsisting of: (a) amino acid residues 218-240 of SEQ ID NO:3 (SEQ IDNO:20) or amino acid residues of a C1q protein chain C (C1qC)corresponding to amino acid residues 218-240 (WLAVNDYYDMVGI QGSDSVFSGF)of SEQ ID NO:3 (SEQ ID NO:20); (b) amino acid residues 225-240 of SEQ IDNO:3 (SEQ ID NO:21) or amino acid residues of a C1qC corresponding toamino acid residues 225-240 (YDMVGI QGSDSVFSGF) of SEQ ID NO:3 (SEQ IDNO:21); (c) amino acid residues 225-232 of SEQ ID NO:3 (SEQ ID NO:22) oramino acid residues of a C1qC corresponding to amino acid residues225-232 (YDMVGIQG) of SEQ ID NO:3 (SEQ ID NO:22); (d) amino acid residueTyr 225 of SEQ ID NO:3 or an amino acid residue of a C1qC correspondingto amino acid residue Tyr 225 of SEQ ID NO:3; (e) amino acid residues174-196 of SEQ ID NO:3 (SEQ ID NO:23) or amino acid residues of a C1qCcorresponding to amino acid residues 174-196 (HTANLCVLLYRSGVKVVTFCGHT)of SEQ ID NO:3 (SEQ ID NO:23); (f) amino acid residues 184-192 of SEQ IDNO:3 (SEQ ID NO:24) or amino acid residues of a C1qC corresponding toamino acid residues 184-192 (RSGVKVVTF) of SEQ ID NO:3 (SEQ ID NO:24);(g) amino acid residues 185-187 of SEQ ID NO:3 or amino acid residues ofa C1qC corresponding to amino acid residues 185-187 (SGV) of SEQ IDNO:3; and (h) amino acid residue Ser 185 of SEQ ID NO:3 or an amino acidresidue of a C1qC corresponding to amino acid residue Ser 185 of SEQ IDNO:3. In certain embodiments, the antibody is a humanized antibody, achimeric antibody, or a human antibody. In certain embodiments, theantibody binds to amino acid residue Lys 219 and Ser 202 of the humanC1qA as shown in SEQ ID NO:1 or amino acids of a human C1qAcorresponding to Lys 219 and Ser 202 as shown in SEQ ID NO:1, and aminoacid residue Tyr 225 of the human C1qC as shown in SEQ ID NO:3 or anamino acid residue of a human C1qC corresponding to Tyr 225 as shown inSEQ ID NO:3. In certain embodiments, the antibody binds to amino acidresidue Lys 219 of the human C1qA as shown in SEQ ID NO:1 or an aminoacid residue of a human C1qA corresponding to Lys 219 as shown in SEQ IDNO:1, and amino acid residue Ser 185 of the human C1qC as shown in SEQID NO:3 or an amino acid residue of a human C1qC corresponding to Ser185 as shown in SEQ ID NO:3.

In certain aspects, the present disclosure provides an isolated anti-C1qantibody, which binds to a C1q protein and binds to one or more aminoacids of the C1q protein within amino acid residues selected from thegroup consisting of: (a) amino acid residues 218-240 of SEQ ID NO:3 (SEQID NO:20) or amino acid residues of a C1qC corresponding to amino acidresidues 218-240 (WLAVNDYYDMVGI QGSDSVFSGF) of SEQ ID NO:3 (SEQ IDNO:20); (b) amino acid residues 225-240 of SEQ ID NO:3 (SEQ ID NO:21) oramino acid residues of a C1qC corresponding to amino acid residues225-240 (YDMVGI QGSDSVFSGF) of SEQ ID NO:3 (SEQ ID NO:21); (c) aminoacid residues 225-232 of SEQ ID NO:3 (SEQ ID NO:22) or amino acidresidues of a C1qC corresponding to amino acid residues 225-232(YDMVGIQG) of SEQ ID NO:3 (SEQ ID NO:22); (d) amino acid residue Tyr 225of SEQ ID NO:3 or an amino acid residue of a C1qC corresponding to aminoacid residue Tyr 225 of SEQ ID NO:3; (e)amino acid residues 174-196 ofSEQ ID NO:3 (SEQ ID NO:23) or amino acid residues of a C1qCcorresponding to amino acid residues 174-196 (HTANLCVLLYRSGVKVVTFCGHT)of SEQ ID NO:3 (SEQ ID NO:23); (f) amino acid residues 184-192 of SEQ IDNO:3 (SEQ ID NO:24) or amino acid residues of a C1qC corresponding toamino acid residues 184-192 (RSGVKVVTF) of SEQ ID NO:3 (SEQ ID NO:24);(g) amino acid residues 185-187 of SEQ ID NO:3 or amino acid residues ofa C1qC corresponding to amino acid residues 185-187 (SGV) of SEQ IDNO:3; and (h) amino acid residue Ser 185 of SEQ ID NO:3 or an amino acidresidue of a C1qC corresponding to amino acid residue Ser 185 of SEQ IDNO:3. In certain embodiments, the antibody is a humanized antibody, achimeric antibody, or a human antibody. In certain embodiments, theantibody binds to amino acid residue Lys 219 and Ser 202 of the humanC1qA as shown in SEQ ID NO:1 or amino acids of a human C1qAcorresponding to Lys 219 and Ser 202 as shown in SEQ ID NO:1, and aminoacid residue Tyr 225 of the human C1qC as shown in SEQ ID NO:3 or anamino acid residue of a human C1qC corresponding to Tyr 225 as shown inSEQ ID NO:3. In certain embodiments, the antibody binds to amino acidresidue Lys 219 of the human C1qA as shown in SEQ ID NO:1 or an aminoacid residue of a human C1qA corresponding to Lys 219 as shown in SEQ IDNO:1, and amino acid residue Ser 185 of the human C1qC as shown in SEQID NO:3 or an amino acid residue of a human C1qC corresponding to Ser185 as shown in SEQ ID NO:3.

In some embodiments that may be combined with any of the precedingembodiments, the antibody is a humanized antibody, a chimeric antibody,or a human antibody. In some embodiments that may be combined with anyof the preceding embodiments, the antibody binds specifically to bothhuman C1q and mouse C1q. In some embodiments that may be combined withany of the preceding embodiments, the antibody binds specifically to ratC1q. In some embodiments that may be combined with any of the precedingembodiments, the antibody binds specifically to human C1q, mouse C1q,and rat C1q. In some embodiments that may be combined with any of thepreceding embodiments, the antibody has dissociation constant (K_(D))for human C1q and mouse C1q that ranges from less than about 30 nM toless than about 100 pM. In some embodiments that may be combined withany of the preceding embodiments, the antibody has dissociation constant(K_(D)) for human C1q and mouse C1q of less than about 30 nM. In someembodiments that may be combined with any of the preceding embodiments,the antibody has dissociation constant (K_(D)) for human C1q and mouseC1q of less than about 20 nM. In some embodiments that may be combinedwith any of the preceding embodiments, the antibody has dissociationconstant (K_(D)) for human C1q and mouse C1q of less than about 10 nM.In some embodiments that may be combined with any of the precedingembodiments, the antibody has dissociation constant (K_(D)) for humanC1q and mouse C1q of less than about 5 nM. In some embodiments that maybe combined with any of the preceding embodiments, the antibody hasdissociation constant (K_(D)) for human C1q and mouse C1q of less thanabout 1 nM. In some embodiments that may be combined with any of thepreceding embodiments, the antibody has dissociation constants (K_(D))for human C1q and mouse C1q of less than 100 pM or less than about 100pM. In some embodiments that may be combined with any of the precedingembodiments, the antibody specifically binds to and neutralizes abiological activity of C1q. In some embodiments that may be combinedwith any of the preceding embodiments, the biological activity is (1)C1q binding to an autoantibody, (2) C1q binding to C1r, (3) C1q bindingto C1s, (4) C1q binding to phosphatidylserine, (5) C1q binding topentraxin-3, (6) C1q binding to C-reactive protein (CRP), (7) C1qbinding to globular C1q receptor (gC1qR), (8) C1q binding to complementreceptor 1 (CR1), (9) C1q binding to beta-amyloid, or (10) C1q bindingto calreticulin. In some embodiments that may be combined with any ofthe preceding embodiments, the biological activity is (1) activation ofthe classical complement activation pathway, (2) activation of antibodyand complement dependent cytotoxicity, (3) CH50 hemolysis, (4) synapseloss, (5) B-cell antibody production, (6) dendritic cell maturation, (7)T-cell proliferation, (8) cytokine production (9) microglia activation,(10) Arthus reaction, (11) phagocytosis of synapses or nerve endings, or(12) activation of complement receptor 3 (CR3/C3) expressing cells. Insome embodiments that may be combined with any of the precedingembodiments, CH50 hemolysis comprises human, mouse, and/or rat CH50hemolysis. In some embodiments that may be combined with any of thepreceding embodiments, the antibody is capable of neutralizing at least50%, at least 80%, or at least 90% of CH50 hemolysis. In someembodiments that may be combined with any of the preceding embodiments,the antibody is capable of neutralizing at least 50% of CH50 hemolysisat a dose of less than 200 ng/ml, less than 100 ng/ml, less than 50ng/ml, or less than 20 ng/ml.

In some embodiments that may be combined with any of the precedingembodiments, the antibody is a bispecific antibody. In some embodimentsthat may be combined with any of the preceding embodiments, the antibodyhas been engineered to increase brain penetration. In some embodimentsthat may be combined with any of the preceding embodiments, the antibodyis a bispecific antibody recognizing a first antigen and a secondantigen. In some embodiments that may be combined with any of thepreceding embodiments, the first antigen is a C1q protein and the secondantigen is an antigen facilitating transport across theblood-brain-barrier. In some embodiments that may be combined with anyof the preceding embodiments, the second antigen is selected from thegroup consisting of transferrin receptor (TR), insulin receptor (HIR),insulin-like growth factor receptor (IGFR), low-density lipoproteinreceptor related proteins 1 and 2 (LPR-1 and 2), diphtheria toxinreceptor, CRM197, a llama single domain antibody, TMEM 30(A), a proteintransduction domain, TAT, Syn-B, penetratin, a poly-arginine peptide, anangiopep peptide, and ANG1005. In some embodiments that may be combinedwith any of the preceding embodiments, the antibody is of the IgG class.In some embodiments that may be combined with any of the precedingembodiments, the antibody has an IgG₁, IgG₂, IgG₃, or IgG₄ isotype. Insome embodiments that may be combined with any of the precedingembodiments, the antibody is an antibody fragment. In some embodimentsthat may be combined with any of the preceding embodiments, the antibodyis a Fab, F(ab′)₂, or Fab′ fragment. In some embodiments that may becombined with any of the preceding embodiments, the antibody fragmentspecifically binds to and neutralizes a biological activity of C1q. Insome embodiments that may be combined with any of the precedingembodiments, the antibody fragment has better brain penetration ascompared to its corresponding full-length antibody. In some embodimentsthat may be combined with any of the preceding embodiments, the antibodyfragment has a shorter half-life as compared to its correspondingfull-length antibody.

In certain aspects, the present disclosure provides for an isolatedpolynucleotide comprising a nucleic acid sequence encoding an antibodyof this disclosure. In certain aspects, the present disclosure providesfor an isolated polynucleotide comprising a nucleic acid sequenceencoding an anti-C1q antibody of any of the preceding embodiments. Incertain aspects, the present disclosure provides for an isolated hostcell comprising a nucleic acid sequence of this disclosure. In certainaspects, the present disclosure provides for an isolated host cellcomprising a nucleic acid sequence of any of the preceding embodiments.In certain aspects, the present disclosure provides for a hybridoma celldeposited with ATCC Accession Number PTA-120399, or progeny thereof. Incertain aspects, the present disclosure provides for a pharmaceuticalcomposition comprising an antibody of this disclosure and apharmaceutically acceptable carrier. In certain aspects, the presentdisclosure provides for a pharmaceutical composition comprising ananti-C1q antibody of any of the preceding embodiments and apharmaceutically acceptable carrier.

In certain aspects, the present disclosure provides for a method oftreating or preventing a disease associated with complement activationin an individual in need of such treatment, the method comprising thestep of administering a therapeutically effective dose of an antibody ofthis disclosure. In certain aspects, the present disclosure provides fora method of treating or preventing a disease associated with complementactivation in an individual in need of such treatment, the methodcomprising the step of administering a therapeutically effective dose ofan anti-C1q antibody of any of the preceding embodiments. In otheraspects, the present disclosure provides an anti-C1q antibody of any ofthe preceding embodiments for use in treating or preventing a diseaseassociated with complement activation in an individual in need of suchtreatment. In other aspects, the present disclosure provides use of ananti-C1q antibody of any of the preceding embodiments in the manufactureof a medicament for treating or preventing a disease associated withcomplement activation in an individual in need of such treatment.

In some embodiments that may be combined with any of the precedingembodiments, the disease associated with complement activation is aneurodegenerative disorder. In some embodiments that may be combinedwith any of the preceding embodiments, the neurodegenerative disorder isassociated with the loss of synapses or nerve connections. In someembodiments that may be combined with any of the preceding embodiments,the neurodegenerative disorder is associated with synapse loss that isdependent on the complement receptor 3(CR3)/C3 or complement receptorCR1. In some embodiments that may be combined with any of the precedingembodiments, the neurodegenerative disorder is associated withpathological activity-dependent synaptic pruning. In some embodimentsthat may be combined with any of the preceding embodiments, theneurodegenerative disorder is associated with synapse phagocytosis bymicroglia. In some embodiments that may be combined with any of thepreceding embodiments, the neurodegenerative disorder is Alzheimer'sdisease amyotrophic lateral sclerosis, multiple sclerosis, glaucoma,myotonic dystrophy, Down syndrome, Parkinson's disease, or Huntington'sdisease. In some embodiments that may be combined with any of thepreceding embodiments, the disease associated with complement activationis an inflammatory disease, an autoimmune disease, or metabolicdisorder. In some embodiments that may be combined with any of thepreceding embodiments, the inflammatory disease, autoimmune disease, ormetabolic disorder is diabetes, obesity, rheumatoid arthritis (RA),acute respiratory distress syndrome (ARDS), remote tissue injury afterischemia and reperfusion, complement activation during cardiopulmonarybypass surgery, dermatomyositis, pemphigus, lupus nephritis andresultant glomerulonephritis and vasculitis, cardiopulmonary bypass,cardioplegia-induced coronary endothelial dysfunction, type IImembranoproliferative glomerulonephritis, IgA nephropathy, acute renalfailure, cryoglobulemia, antiphospholipid syndrome, macular degenerativediseases, age-related macular degeneration (AMD), choroidalneovascularization (CNV), uveitis, diabetic retinopathy,ischemia-related retinopathy, endophthalmitis, intraocular neovasculardisease, diabetic macular edema, pathological myopia, von Hippel-Lindaudisease, histoplasmosis of the eye, Neuromyelitis Optica (NMO), CentralRetinal Vein Occlusion (CRVO), corneal neovascularization, retinalneovascularization, allo-transplantation, hyperacute rejection,hemodialysis, chronic occlusive pulmonary distress syndrome (COPD),asthma, or aspiration pneumonia. In some embodiments that may becombined with any of the preceding embodiments, the disease associatedwith complement activation is an autoimmune disease selected from thegroup consisting of myasthenia gravis, Diabetes mellitus type 1,Hashimoto's thyroiditis, Addison's disease, Coeliac disease, Crohn'sdisease, pernicious anaemia, Pemphigus vulgaris, vitiligo, autoimmunehemolytic anemias, paraneoplastic syndromes, a vasculitis disease,polymyalgia rheumatica, temporal arteritis, and Wegener'sgranulomatosis.

In certain aspects, the present disclosure provides for a kit comprisingan anti-C1q antibody of any of the preceding embodiments, and a packageinsert comprising instructions for using the antibody to treat orprevent a disease associated with complement activation in an individualin need of such treatment. In some embodiments, the disease associatedwith complement activation is a neurodegenerative disorder. In someembodiments, the neurodegenerative disorder is associated with loss ofsynapses or loss nerve connections. In some embodiments, theneurodegenerative disorder is associated with synapse loss that isdependent on the complement receptor 3(CR3)/C3 or complement receptorCR1. In some embodiments, the neurodegenerative disorder is associatedwith pathological activity-dependent synaptic pruning. In someembodiments, the neurodegenerative disorder is associated with synapsephagocytosis by microglia. In some embodiments, the neurodegenerativedisorder is selected from the group consisting of Alzheimer's disease,amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonicdystrophy, Down syndrome, Parkinson's disease, and Huntington's disease.In some embodiments, the disease associated with complement activationis an inflammatory disease, autoimmune disease, or metabolic disorder.In some embodiments, the inflammatory disease, autoimmune disease, ormetabolic disorder is selected from the group consisting of diabetes,obesity, rheumatoid arthritis (RA), acute respiratory distress syndrome(ARDS), remote tissue injury after ischemia and reperfusion, complementactivation during cardiopulmonary bypass surgery, dermatomyositis,pemphigus, lupus nephritis and resultant glomerulonephritis andvasculitis, cardiopulmonary bypass, cardioplegia-induced coronaryendothelial dysfunction, type II membranoproliferativeglomerulonephritis, IgA nephropathy, acute renal failure,cryoglobulemia, antiphospholipid syndrome, macular degenerativediseases, age-related macular degeneration (AMD), choroidalneovascularization (CNV), uveitis, diabetic retinopathy,ischemia-related retinopathy, endophthalmitis, intraocular neovasculardisease, diabetic macular edema, pathological myopia, von Hippel-Lindaudisease, histoplasmosis of the eye, Neuromyelitis Optica (NMO), CentralRetinal Vein Occlusion (CRVO), corneal neovascularization, retinalneovascularization, allo-transplantation, hyperacute rejection,hemodialysis, chronic occlusive pulmonary distress syndrome (COPD),asthma, and aspiration pneumonia. In some embodiments, the diseaseassociated with complement activation is an autoimmune disease selectedfrom the group consisting of myasthenia gravis, Diabetes mellitus type1, Hashimoto's thyroiditis, Addison's disease, Coeliac disease, Crohn'sdisease, pernicious anaemia, Pemphigus vulgaris, vitiligo, autoimmunehemolytic anemias, paraneoplastic syndromes, a vasculitis disease,polymyalgia rheumatica, temporal arteritis, and Wegener'sgranulomatosis.

In certain aspects, the present disclosure provides for a diagnostic kitcomprising an antibody of this disclosure. In certain aspects, thepresent disclosure provides for a kit comprising an anti-C1q antibody ofany of the preceding embodiments. In some embodiments, the kit is fordiagnostic or therapeutic uses as disclosed herein.

In certain aspects, the present disclosure provides for a method ofdetecting synapses in an individual having a neurodegenerative diseaseor autoimmune disease, the method comprising a) administering anantibody of this disclosure to the individual, and b) detecting antibodybound to synapses, thereby detecting synapses in the individual. Inother aspects, the present disclosure provides a method of detectingsynapses in an individual, by a) administering an anti-C1q antibody ofany of the preceding embodiments to the individual, and b) detectingantibody bound to synapses, thereby detecting synapses in theindividual. In other aspects, the present disclosure provides ananti-C1q antibody of any of the preceding embodiments for use indetecting synapses in an individual. In other aspects, the presentdisclosure provides use of an anti-C1q antibody of any of the precedingembodiments in the manufacture of a medicament for detecting synapses inan individual. In some embodiments that may be combined with any of thepreceding embodiments, the antibody bound to synapses is detected usingimaging techniques selected from the group consisting of positronemission tomography (PET), X-ray computed tomography, single-photonemission computed tomography (SPECT), computed tomography (CT), andcomputed axial tomography (CAT). In some embodiments that may becombined with any of the preceding embodiments, the detection ofantibody bound to synapses provides a quantitative measure of the numberof synapses in the individual. In some embodiments that may be combinedwith any of the preceding embodiments, the individual has aneurodegenerative disease or autoimmune disease. In some embodimentsthat may be combined with any of the preceding embodiments, the numberof synapses in the individual is measured repeatedly over a period oftime and a loss of synapses in the individual is detected over time. Insome embodiments that may be combined with any of the precedingembodiments, the loss of synapses over time is a measure for theefficacy of a treatment for the neurodegenerative disease or autoimmunedisease.

In certain aspects, the present disclosure provides for a method ofdetecting synapses in a biological sample, the method comprising a)contacting the biological sample with an antibody of this disclosure andb) detecting antibody bound to synapses, thereby detecting synapses inthe biological sample. In certain aspects, the present disclosureprovides for a method of detecting synapses in a biological sample, themethod comprising a) contacting the biological sample with an antibodyof this disclosure and b) detecting antibody bound to synapses, therebydetecting synapses in the biological sample. In other aspects, thepresent disclosure provides a method of detecting synapses in abiological sample, by a) contacting the biological sample with ananti-C1q antibody of any of the preceding embodiments, and b) detectingantibody bound to synapses, thereby detecting synapses in theindividual.

In some embodiments that may be combined with any of the precedingembodiments, the method further comprises a step before step a) ofobtaining the biological sample from an individual. In some embodimentsthat may be combined with any of the preceding embodiments, thebiological sample comprises a biopsy specimen, a tissue, or a cell. Insome embodiments that may be combined with any of the precedingembodiments, the antibody is detected by immunofluorescence microscopy,immunocytochemistry, immunohistochemistry, ELISA, FACS analysis orimmunoprecipitation.

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be combined to form otherembodiments of the compositions and methods provided herein. These andother aspects of the compositions and methods provided herein willbecome apparent to one of skill in the art.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the results of an ELISA screen for antibodiesspecifically binding human C1q. Hybridoma supernatants containinganti-C1q antibodies 1C7, 2A1, 3A2, or 5A3 respectively were tested. Leftcolumns (grey) represent signals for anti-C1q antibody-binding to humanC1q protein. Right columns (black) represent signals for anti-C1qantibody-binding to human transferrin (HT).

FIG. 2 illustrates the C1q-neutralizing activities of anti-C1qantibodies 1C7, 2A1, 3A2, and 5A3 in a human CH50 hemolytic assay in asingle-dose format.

FIG. 3 illustrates the C1q-neutralizing activities of anti-C1qantibodies 1C7, 3A2, and 4A4B11 in a human CH50 hemolytic assay in adose-response format.

FIG. 4A, FIG. 4B, and FIG. 4C illustrate the C1q-neutralizing activitiesof anti-C1q antibodies M1 and 4A4B11 in human, mouse, and rat CH50hemolytic assays in a dose-response format. FIG. 4A illustrates resultsfrom a human CH50 hemolytic assay. FIG. 4B illustrates results from amouse CH50 hemolytic assay. FIG. 4C illustrates results from a rat CH50hemolytic assay.

FIG. 5A and FIG. 5B illustrate mass spectrometry characterization of C1qantibody complexes. FIG. 5A shows a mixture of ANN-001 (4A4B11) and C1qshows that ANN-001 monomer at the predicted mass of ˜150 kDa, C1qmonomer at the expected mass of ˜460 kDa, and the C1q/ANN-001 1:1complex at the predicted mass of ˜600 kDa. FIG. 5B shows a mixture ofANN-005 (M1) and C1q shows that ANN-005 monomer at the predicted mass of˜150 kDa, C1q monomer at the expected mass of ˜460 kDa, and theC1q/ANN-005 1:1 complex at the predicted mass of ˜600 kDa.

FIG. 6A and FIG. 6B illustrate C1q peptides do not compete with intactC1q for binding to monoclonal antibody ANN-005 (M1). FIG. 6A depicts C1qand ANN-005 mixed in equimolar concentrations and incubated in theabsence of a mixture of C1q peptides. FIG. 6B depicts C1q and ANN-005mixed in equimolar concentrations and incubated in the presence of amixture of C1q peptides generated by pepsin digestion of C1q andanalyzed by mass spectrometry. In each case, a portion of the unboundantibody and antigen (ANN-005 and C1q) can be identified at the expectedmasses for monomers (˜150 kDa and ˜460 kDa respectively) and a 1:1complex is present at a mass of ˜615 kDa.

DETAILED DESCRIPTION OF THE INVENTION

General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3d edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Current Protocols inMolecular Biology (F. M. Ausubel, et al. eds., (2003)); the seriesMethods in Enzymology (Academic Press, Inc.): PCR 2: A PracticalApproach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and AnimalCell Culture (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press;Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Celland Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B.Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbookof Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos,eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.,1994); Current Protocols in Immunology (J. E. Coligan et al., eds.,1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D. Catty, ed., IRLPress, 1988-1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); UsingAntibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D.Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principlesand Practice of Oncology (V. T. DeVita et al., eds., J.B. LippincottCompany, 1993).

Definitions

As used herein, the term “preventing” includes providing prophylaxiswith respect to occurrence or recurrence of a particular disease,disorder, or condition in an individual. An individual may bepredisposed to, susceptible to a particular disease, disorder, orcondition, or at risk of developing such a disease, disorder, orcondition, but has not yet been diagnosed with the disease, disorder, orcondition.

As used herein, an individual “at risk” of developing a particulardisease, disorder, or condition may or may not have detectable diseaseor symptoms of disease, and may or may not have displayed detectabledisease or symptoms of disease prior to the treatment methods describedherein. “At risk” denotes that an individual has one or more riskfactors, which are measurable parameters that correlate with developmentof a particular disease, disorder, or condition, as known in the art. Anindividual having one or more of these risk factors has a higherprobability of developing a particular disease, disorder, or conditionthan an individual without one or more of these risk factors.

As used herein, the term “treatment” refers to clinical interventiondesigned to alter the natural course of the individual being treatedduring the course of clinical pathology. Desirable effects of treatmentinclude decreasing the rate of progression, ameliorating or palliatingthe pathological state, and remission or improved prognosis of aparticular disease, disorder, or condition. An individual issuccessfully “treated”, for example, if one or more symptoms associatedwith a particular disease, disorder, or condition are mitigated oreliminated.

An “effective amount” refers to at least an amount effective, at dosagesand for periods of time necessary, to achieve the desired therapeutic orprophylactic result. An effective amount can be provided in one or moreadministrations.

A “therapeutically effective amount” is at least the minimumconcentration required to effect a measurable improvement of aparticular disease, disorder, or condition. A therapeutically effectiveamount herein may vary according to factors such as the disease state,age, sex, and weight of the patient, and the ability of the anti-C1qantibody to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the anti-C1q antibody are outweighed by thetherapeutically beneficial effects.

Chronic” administration refers to administration of the medicament(s) ina continuous as opposed to acute mode, so as to maintain the initialtherapeutic effect (activity) for an extended period of time.“Intermittent” administration refers to treatment that is notconsecutively done without interruption, but rather is cyclic in nature.

As used herein, administration “in conjunction” with another compound orcomposition includes simultaneous administration and/or administrationat different times. Administration in conjunction also encompassesadministration as a co-formulation or administration as separatecompositions, including at different dosing frequencies or intervals,and using the same route of administration or different routes ofadministration.

An “individual” for purposes of treatment, prevention, or reduction ofrisk refers to any animal classified as a mammal, including humans,domestic and farm animals, and zoo, sport, or pet animals, such as dogs,horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats,cats, and the like. In some embodiments, the individual is human.

As used herein, “autoantibody” means any antibody that recognizes a hostantigen.

The term “immunoglobulin” (Ig) is used interchangeably with “antibody”herein. The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired biological activity.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains. The pairing of a V_(H) and V_(L) together forms a singleantigen-binding site. For the structure and properties of the differentclasses of antibodies, see, e.g., Basic and Clinical Immunology, 8thEd., Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.),Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa (“κ”) and lambda (“λ”), based onthe amino acid sequences of their constant domains. Depending on theamino acid sequence of the constant domain of their heavy chains (CH),immunoglobulins can be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated alpha (“α”), delta (“δ”), epsilon (“ε”), gamma(“γ”) and mu (“μ”), respectively. The γ and α classes are furtherdivided into subclasses (isotypes) on the basis of relatively minordifferences in the CH sequence and function, e.g., humans express thefollowing subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Thesubunit structures and three dimensional configurations of differentclasses of immunoglobulins are well known and described generally in,for example, Abbas et al., Cellular and Molecular Immunology, 4^(th) ed.(W.B. Saunders Co., 2000).

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains.

An “isolated” antibody, such as an anti-C1q antibody of the presentdisclosure, is one that has been identified, separated and/or recoveredfrom a component of its production environment (e.g., naturally orrecombinantly). In some embodiments, the isolated polypeptide is free ofassociation with all other contaminant components from its productionenvironment. Contaminant components from its production environment,such as those resulting from recombinant transfected cells, arematerials that would typically interfere with research, diagnostic ortherapeutic uses for the antibody, and may include enzymes, hormones,and other proteinaceous or non-proteinaceous solutes. In someembodiments, the polypeptide will be purified: (1) to greater than 95%by weight of antibody as determined by, for example, the Lowry method,and in some embodiments, to greater than 99% by weight; (2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or (3) tohomogeneity by SDS-PAGE under non-reducing or reducing conditions usingCoomassie blue or silver stain. Isolated antibody includes the antibodyin situ within recombinant T-cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,an isolated polypeptide or antibody will be prepared by at least onepurification step.

The “variable region” or “variable domain” of an antibody, such as ananti-C1q antibody of the present disclosure, refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domains of the heavy chain and light chain may be referred toas “V_(H)” and “V_(L)”, respectively. These domains are generally themost variable parts of the antibody (relative to other antibodies of thesame class) and contain the antigen binding sites.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies, suchas anti-C1q antibodies of the present disclosure. The V domain mediatesantigen binding and defines the specificity of a particular antibody forits particular antigen. However, the variability is not evenlydistributed across the entire span of the variable domains. Instead, itis concentrated in three segments called hypervariable regions (HVRs)both in the light-chain and the heavy chain variable domains. The morehighly conserved portions of variable domains are called the frameworkregions (FR). The variable domains of native heavy and light chains eachcomprise four FR regions, largely adopting a beta-sheet configuration,connected by three HVRs, which form loops connecting, and in some casesforming part of, the beta-sheet structure. The HVRs in each chain areheld together in close proximity by the FR regions and, with the HVRsfrom the other chain, contribute to the formation of the antigen bindingsite of antibodies (see Kabat et al., Sequences of ImmunologicalInterest, Fifth Edition, National Institute of Health, Bethesda, Md.(1991)). The constant domains are not involved directly in the bindingof antibody to an antigen, but exhibit various effector functions, suchas participation of the antibody in antibody-dependent-cellulartoxicity.

The term “monoclonal antibody” as used herein refers to an antibody,such as an anti-C1q antibody of the present disclosure, obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies comprising the population are identical except for possiblenaturally occurring mutations and/or post-translation modifications(e.g., isomerizations, amidations) that may be present in minor amounts.Monoclonal antibodies are highly specific, being directed against asingle antigenic site. In contrast to polyclonal antibody preparationswhich typically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody is directed against asingle determinant on the antigen. In addition to their specificity, themonoclonal antibodies are advantageous in that they are synthesized bythe hybridoma culture, uncontaminated by other immunoglobulins. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by a variety oftechniques, including, for example, the hybridoma method (e.g., Kohlerand Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual,(Cold Spring Harbor Laboratory Press, 2d ed. 1988); Hammerling et al.,in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567),phage-display technologies (see, e.g., Clackson et al., Nature,352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992);Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol.Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Nat'l Acad. Sci. USA101(34):12467-472 (2004); and Lee et al., J. Immunol. Methods284(1-2):119-132 (2004), and technologies for producing human orhuman-like antibodies in animals that have parts or all of the humanimmunoglobulin loci or genes encoding human immunoglobulin sequences(see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741;Jakobovits et al., Proc. Nat'l Acad. Sci. USA 90:2551 (1993); Jakobovitset al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol.7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; and 5,661,016; Marks et al., Bio/Technology 10:779-783(1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851(1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg andHuszar, Intern. Rev. Immunol. 13:65-93 (1995).

The terms “full-length antibody,” “intact antibody” or “whole antibody”are used interchangeably to refer to an antibody, such as and anti-C1qantibody of the present disclosure, in its substantially intact form, asopposed to an antibody fragment. Specifically whole antibodies includethose with heavy and light chains including an Fc region. The constantdomains may be native sequence constant domains (e.g., human nativesequence constant domains) or amino acid sequence variants thereof. Insome cases, the intact antibody may have one or more effector functions.

An “antibody fragment” comprises a portion of an intact antibody, theantigen binding and/or the variable region of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)₂ and Fvfragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 (1995));single-chain antibody molecules and multispecific antibodies formed fromantibody fragments.

Papain digestion of antibodies, such as anti-C1q antibodies of thepresent disclosure, produces two identical antigen-binding fragments,called “Fab” fragments, and a residual “Fc” fragment, a designationreflecting the ability to crystallize readily. The Fab fragment consistsof an entire L chain along with the variable region domain of the Hchain (V_(H)), and the first constant domain of one heavy chain(C_(H)1). Each Fab fragment is monovalent with respect to antigenbinding, i.e., it has a single antigen-binding site. Pepsin treatment ofan antibody yields a single large F(ab′)₂ fragment which roughlycorresponds to two disulfide linked Fab fragments having differentantigen-binding activity and is still capable of cross-linking antigen.Fab′ fragments differ from Fab fragments by having a few additionalresidues at the carboxy terminus of the C_(H)1 domain including one ormore cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, the region which is alsorecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the VH and VL antibody domains connected into asingle polypeptide chain. In some embodiments, the sFv polypeptidefurther comprises a polypeptide linker between the V_(H) and V_(L)domains which enables the sFv to form the desired structure for antigenbinding. For a review of the sFv, see Plückthun in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269-315 (1994).

“Functional fragments” of antibodies, such as anti-C1q antibodies of thepresent disclosure, comprise a portion of an intact antibody, generallyincluding the antigen binding or variable region of the intact antibodyor the F region of an antibody which retains or has modified FcR bindingcapability. Examples of antibody fragments include linear antibody,single-chain antibody molecules and multispecific antibodies formed fromantibody fragments.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10) residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,thereby resulting in a bivalent fragment, i.e., a fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described in greater detail in, for example, EP 404,097; WO93/11161; Hollinger et al., Proc. Nat'l Acad. Sci. USA 90:6444-48(1993).

As used herein, a “chimeric antibody” refers to an antibody(immunoglobulin), such as an anti-C1q antibody of the presentdisclosure, in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is(are) identicalwith or homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; Morrison et al.,Proc. Nat'l Acad. Sci. USA, 81:6851-55 (1984)). Chimeric antibodies ofinterest herein include PRIMATIZED® antibodies wherein theantigen-binding region of the antibody is derived from an antibodyproduced by, e.g., immunizing macaque monkeys with an antigen ofinterest. As used herein, “humanized antibody” is used a subset of“chimeric antibodies.”

“Humanized” forms of non-human (e.g., murine) antibodies, such asanti-C1q antibodies of the present disclosure, are chimeric antibodiesthat contain minimal sequence derived from non-human immunoglobulin. Inone embodiment, a humanized antibody is a human immunoglobulin(recipient antibody) in which residues from an HVR of the recipient arereplaced by residues from an HVR of a non-human species (donor antibody)such as mouse, rat, rabbit or non-human primate having the desiredspecificity, affinity, and/or capacity. In some instances, FR residuesof the human immunoglobulin are replaced by corresponding non-humanresidues. Furthermore, humanized antibodies may comprise residues thatare not found in the recipient antibody or in the donor antibody. Thesemodifications may be made to further refine antibody performance, suchas binding affinity. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin sequence, and all orsubstantially all of the FR regions are those of a human immunoglobulinsequence, although the FR regions may include one or more individual FRresidue substitutions that improve antibody performance, such as bindingaffinity, isomerization, immunogenicity, and the like. The number ofthese amino acid substitutions in the FR is typically no more than 6 inthe H chain, and in the L chain, no more than 3. The humanized antibodyoptionally will also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see, e.g., Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op.Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani andHamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris,Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr.Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and7,087,409.

A “human antibody” is one that possesses an amino-acid sequencecorresponding to that of an antibody, such as an anti-C1q antibody ofthe present disclosure, produced by a human and/or has been made usingany of the techniques for making human antibodies as disclosed herein.This definition of a human antibody specifically excludes a humanizedantibody comprising non-human antigen-binding residues. Human antibodiescan be produced using various techniques known in the art, includingphage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381(1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available forthe preparation of human monoclonal antibodies are methods described inCole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See alsovan Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001).Human antibodies can be prepared by administering the antigen to atransgenic animal that has been modified to produce such antibodies inresponse to antigenic challenge, but whose endogenous loci have beendisabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181and 6,150,584 regarding XENOMOUSE™ technology). See also, for example,Li et al., Proc. Nat'l Acad. Sci. USA, 103:3557-3562 (2006) regardinghuman antibodies generated via a human B-cell hybridoma technology.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody-variable domain, such as that of ananti-C1q antibody of the present disclosure, that are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al.,Immunity 13:37-45 (2000); Johnson and Wu in Methods in Molecular Biology248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003)). Indeed, naturallyoccurring camelid antibodies consisting of a heavy chain only arefunctional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheHVRs that are Kabat complementarity-determining regions (CDRs) are basedon sequence variability and are the most commonly used (Kabat et al.,supra). Chothia refers instead to the location of the structural loops(Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM HVRsrepresent a compromise between the Kabat CDRs and Chothia structuralloops, and are used by Oxford Molecular's AbM antibody-modelingsoftware. The “contact” HVRs are based on an analysis of the availablecomplex crystal structures. The residues from each of these HVRs arenoted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 (H1), 50-65or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in the VH. Thevariable-domain residues are numbered according to Kabat et al., supra,for each of these extended-HVR definitions.

“Framework” or “FR” residues are those variable-domain residues otherthan the HVR residues as herein defined.

The phrase “variable-domain residue-numbering as in Kabat” or“amino-acid-position numbering as in Kabat,” and variations thereof,refers to the numbering system used for heavy-chain variable domains orlight-chain variable domains of the compilation of antibodies in Kabatet al., supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy-chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g., residues 82a, 82b, and 82c, etc. according toKabat) after heavy-chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). The “EU numbering system”or “EU index” is generally used when referring to a residue in animmunoglobulin heavy chain constant region (e.g., the EU index reportedin Kabat et al., supra). The “EU index as in Kabat” refers to theresidue numbering of the human IgG1 EU antibody. Unless stated otherwiseherein, references to residue numbers in the variable domain ofantibodies means residue numbering by the Kabat numbering system. Unlessstated otherwise herein, references to residue numbers in the constantdomain of antibodies means residue numbering by the EU numbering system(e.g., see United States Patent Publication No. 2010-280227).

An “acceptor human framework” as used herein is a framework comprisingthe amino acid sequence of a VL or VH framework derived from a humanimmunoglobulin framework or a human consensus framework. An acceptorhuman framework “derived from” a human immunoglobulin framework or ahuman consensus framework may comprise the same amino acid sequencethereof, or it may contain pre-existing amino acid sequence changes. Insome embodiments, the number of pre-existing amino acid changes are 10or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 orless, 3 or less, or 2 or less. In some embodiments, where pre-existingamino acid changes are present in a VH, those changes occur at onlythree, two, or one of positions 71H, 73H and 78H; for instance, theamino acid residues at those positions may by 71A, 73T and/or 78A. Inone embodiment, the VL acceptor human framework is identical in sequenceto the VL human immunoglobulin framework sequence or human consensusframework sequence.

A “human consensus framework” is a framework that represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md.(1991). Examples include for the VL, the subgroup may be subgroup kappaI, kappa II, kappa III or kappa IV as in Kabat et al., supra.Additionally, for the VH, the subgroup may be subgroup I, subgroup II,or subgroup III as in Kabat et al., supra.

An “amino-acid modification” at a specified position, e.g., of ananti-C1q antibody of the present disclosure, refers to the substitutionor deletion of the specified residue, or the insertion of at least oneamino acid residue adjacent the specified residue. Insertion “adjacent”to a specified residue means insertion within one to two residuesthereof. The insertion may be N-terminal or C-terminal to the specifiedresidue. In some embodiments, the amino acid modification herein is asubstitution.

An “affinity-matured” antibody, such as an anti-C1q antibody of thepresent disclosure, is one with one or more alterations in one or moreHVRs thereof that result in an improvement in the affinity of theantibody for antigen, compared to a parent antibody that does notpossess those alteration(s). In one embodiment, an affinity-maturedantibody has nanomolar or even picomolar affinities for the targetantigen. Affinity-matured antibodies are produced by procedures known inthe art. For example, Marks et al., Bio/Technology 10:779-783 (1992)describes affinity maturation by VH- and VL-domain shuffling. Randommutagenesis of HVR and/or framework residues is described by, forexample: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994);Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol.155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995);and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

As use herein, the term “specifically recognizes” or “specificallybinds” refers to measurable and reproducible interactions such asattraction or binding between a target and an antibody, such as ananti-C1q antibody of the present disclosure, that is determinative ofthe presence of the target in the presence of a heterogeneous populationof molecules including biological molecules. For example, an antibody,such as an anti-C1q antibody of the present disclosure, thatspecifically or preferentially binds to a target or an epitope is anantibody that binds this target or epitope with greater affinity,avidity, more readily, and/or with greater duration than it binds toother targets or other epitopes of the target. It is also understood byreading this definition that, for example, an antibody (or a moiety)that specifically or preferentially binds to a first target may or maynot specifically or preferentially bind to a second target. As such,“specific binding” or “preferential binding” does not necessarilyrequire (although it can include) exclusive binding. An antibody thatspecifically binds to a target may have an association constant of atleast about 10³ M⁻¹ or 10⁴ M⁻¹, sometimes about 10⁵ M⁻¹ or 10⁶ M⁻¹, inother instances about 10⁶ M⁻¹ or 10⁷ M⁻¹, about 10⁸ M⁻¹ to 10⁹ M⁻¹, orabout 10¹⁰ M⁻¹ to 10¹¹ M⁻¹ or higher. A variety of immunoassay formatscan be used to select antibodies specifically immunoreactive with aparticular protein. For example, solid-phase ELISA immunoassays areroutinely used to select monoclonal antibodies specificallyimmunoreactive with a protein. See, e.g., Harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork, for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity.

As used herein, an “interaction” between a complement protein, such ascomplement factor C1q, and a second protein encompasses, withoutlimitation, protein-protein interaction, a physical interaction, achemical interaction, binding, covalent binding, and ionic binding. Asused herein, an antibody “inhibits interaction” between two proteinswhen the antibody disrupts, reduces, or completely eliminates aninteraction between the two proteins. An antibody of the presentdisclosure, or fragment thereof, “inhibits interaction” between twoproteins when the antibody or fragment thereof binds to one of the twoproteins.

A “blocking” antibody, an “antagonist” antibody, an “inhibitory”antibody, or a “neutralizing” antibody is an antibody, such as ananti-C1q antibody of the present disclosure that inhibits or reduces oneor more biological activities of the antigen it binds, such asinteractions with one or more proteins. In some embodiments, blockingantibodies, antagonist antibodies, inhibitory antibodies, or“neutralizing” antibodies substantially or completely inhibit one ormore biological activities or interactions of the antigen.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native-sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy-chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during production orpurification of the antibody, or by recombinantly engineering thenucleic acid encoding a heavy chain of the antibody. Accordingly, acomposition of intact antibodies may comprise antibody populations withall K447 residues removed, antibody populations with no K447 residuesremoved, and antibody populations having a mixture of antibodies withand without the K447 residue. Suitable native-sequence Fc regions foruse in the antibodies of the invention include human IgG1, IgG2, IgG3and IgG4.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification. In some embodiments, the variant Fc region differs inone or more amino acid substitution(s). In some embodiments, the variantFc region has at least one amino acid substitution compared to a nativesequence Fc region or to the Fc region of a parent polypeptide, e.g.from about one to about ten amino acid substitutions, and, in someembodiments, from about one to about five amino acid substitutions in anative sequence Fc region or in the Fc region of the parent polypeptide.The variant Fc region herein will, in some embodiments, possess at leastabout 80% homology with a native sequence Fc region and/or with an Fcregion of a parent polypeptide, and, in some embodiments, at least about90% homology therewith, and, in some embodiments, at least about 95%homology therewith.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. In some embodiments, the FcR is a native sequence humanFcR. Moreover, in some embodiments, a FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and FcγRIII subclasses, including allelic variants and alternativelyspliced forms of these receptors, FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. Activating receptor FcγRIIA contains animmunoreceptor tyrosine-based activation motif (“ITAM”) in itscytoplasmic domain. Inhibiting receptor FcγRIIB contains animmunoreceptor tyrosine-based inhibition motif (“ITIM”) in itscytoplasmic domain. (see, e.g., M. Daëron, Annu. Rev. Immunol.15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev.Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994);and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995). Other FcRs,including those to be identified in the future, are encompassed by theterm “FcR” herein. FcRs can also increase the serum half-life ofantibodies.

Binding to FcRn in vivo and serum half-life of human FcRn high-affinitybinding polypeptides can be assayed, e.g., in transgenic mice ortransfected human cell lines expressing human FcRn, or in primates towhich the polypeptides having a variant Fc region are administered. WO2004/42072 (Presta) describes antibody variants with improved ordiminished binding to FcRs. See also, e.g., Shields et al., J. Biol.Chem. 9(2):6591-6604 (2001).

The term “k_(on)”, as used herein, is intended to refer to the rateconstant for association of an antibody to an antigen.

The term “k_(off)”, as used herein, is intended to refer to the rateconstant for dissociation of an antibody from the antibody/antigencomplex.

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of an antibody-antigen interaction.

As used herein, “percent (%) amino acid sequence identity” and“homology” with respect to a peptide, polypeptide or antibody sequencerefers to the percentage of amino acid residues in a candidate sequencethat are identical with the amino acid residues in the specific peptideor polypeptide sequence, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity,and not considering any conservative substitutions as part of thesequence identity. Alignment for purposes of determining percent aminoacid sequence identity can be achieved in various ways that are withinthe skill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms known in the art needed toachieve maximal alignment over the full length of the sequences beingcompared.

An “isolated” molecule or cell is a molecule or a cell that isidentified and separated from at least one contaminant molecule or cellwith which it is ordinarily associated in the environment in which itwas produced. In some embodiments, the isolated molecule or cell is freeof association with all components associated with the productionenvironment. The isolated molecule or cell is in a form other than inthe form or setting in which it is found in nature. Isolated moleculestherefore are distinguished from molecules existing naturally in cells;isolated cells are distinguished from cells existing naturally intissues, organs, or individuals. In some embodiments, the isolatedmolecule is an anti-C1q antibody of the present disclosure. In otherembodiments, the isolated cell is a host cell or hybridoma cellproducing an anti-C1q antibody of the present disclosure.

An “isolated” nucleic acid molecule encoding an antibody, such as ananti-C1q antibody of the present disclosure, is a nucleic acid moleculethat is identified and separated from at least one contaminant nucleicacid molecule with which it is ordinarily associated in the environmentin which it was produced. In some embodiments, the isolated nucleic acidis free of association with all components associated with theproduction environment. The isolated nucleic acid molecules encoding thepolypeptides and antibodies herein is in a form other than in the formor setting in which it is found in nature. Isolated nucleic acidmolecules therefore are distinguished from nucleic acid encoding thepolypeptides and antibodies herein existing naturally in cells.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA into which additional DNA segments may beligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors,” or simply, “expressionvectors.” In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may comprise modification(s)made after synthesis, such as conjugation to a label. Other types ofmodifications include, for example, “caps,” substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotides(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl-, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such asarabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs, and basic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), (O)NR2 (“amidate”), P(O)R,P(O)OR′, CO, or CH2 (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

A “host cell” includes an individual cell or cell culture that can be orhas been a recipient for vector(s) for incorporation of polynucleotideinserts. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation. A host cell includes cellstransfected in vivo with a polynucleotide(s) of this invention.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptide; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol(PEG), and PLURONICS™.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. For example, reference to an “antibody” is a reference tofrom one to many antibodies, such as molar amounts, and includesequivalents thereof known to those skilled in the art, and so forth.

It is understood that aspect and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

Overview

The present disclosure provides anti-C1q antibodies and uses thereof.The anti-C1q antibodies of this disclosure specifically bind a C1qprotein of this disclosure. In some embodiments, the anti-C1q antibodiesare C1q neutralizing antibodies. In some embodiments, the anti-C1qantibodies of this disclosure may bind to C1 complex.

In certain aspects, the present disclosure provides murine monoclonalantibody M1, which is produced by a hybridoma cell line referred to asmouse hybridoma C1q-M1 7788-1(M) 051613 and which was deposited withATCC on Jun. 6, 2013 with ATCC Accession Number PTA-120399.

In certain aspects, the present disclosure provides an anti-C1q antibodycomprising a light chain variable domain and a heavy chain variabledomain, wherein the light chain variable domain comprises the lightchain variable domain sequence of antibody M1; and/or wherein the heavychain comprises the heavy chain variable domain sequence of antibody M1.

In certain aspects, the present disclosure provides an anti-C1q antibodycomprising a light chain variable domain and a heavy chain variabledomain, wherein the light chain variable domain comprises the HVR-L1,HVR-L2, and HVR-L3 of monoclonal antibody M1 produced by a hybridomacell line deposited at ATCC with ATCC Accession Number PTA-120399 orprogeny thereof and/or wherein the heavy chain variable domain comprisesthe HVR-H1, HVR-H2, and HVR-H3 of monoclonal antibody M1 produced by ahybridoma cell line deposited at ATCC with Accession Number PTA-120399or progeny thereof.

In certain aspects, the present disclosure provides an anti-C1qantibody, which binds essentially the same C1q epitope as (1) antibodyM1 produced by the hybridoma cell line deposited with ATCC on Jun. 6,2013 and having ATCC Accession Number PTA-120399 or progeny thereof, (2)an antigen binding fragment of antibody M1, or (3) an antibodycomprising the HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3 ofantibody M1.

In some embodiments, the anti-C1q antibodies of this disclosureneutralize a biological activity of C1q. Uses for anti-C1q antibodiesinclude, without limitation, the detection of complement factor C1q,e.g., in individuals having a neurodegenerative disorder associated withcomplement factor 1 (CF1)-dependent pathological synapse loss.Additional non-limiting uses include the inhibition of the classicalpathway of complement activation, e.g., in cases where the classicalcomplement pathway is activated by autoantibodies, such as NMO-specificautoantibodies. Further non-limiting uses for anti-C1q antibodiesinclude the diagnosis and treatment of disorders that are associatedwith elevated expression of complement factors, such as C1q, orassociated with the activation of the complement pathway. Such disordersmay include, without limitation, autoimmune disorders, inflammatorydisorders, and neurodegenerative disorders, including neurodegenerativedisorders associated with synapse loss.

In another aspect, the present disclosure provides an isolated nucleicacid molecule encoding an antibody of this disclosure.

The present disclosure also provides isolated host cells containing anucleic acid molecule that encodes an antibody of this disclosure. Insome embodiments, an isolated host cell line is provided that producesthe neutralizing monoclonal murine antibody M1. This isolated host celllines was deposited with ATCC and has ATCC Accession Number PTA-120399.

Additionally, pharmaceutical compositions are provided containinganti-C1q antibodies, such as C1q neutralizing antibodies of thisdisclosure, in combination with pharmaceutically acceptable carriers.The present disclosure also provides a kit containing an anti-C1qantibody for use in any of the methods described herein.

The present disclosure further provides methods of using the C1qantibodies of this disclosure (e.g., C1q neutralizing antibodies of thisdisclosure) to treat or prevent a neurodegenerative disease orautoimmune disease in an individual in need of such treatment, to detectsynapses in an individual having a neurodegenerative disease orautoimmune disease, and to detect synapses in a biological sample. Thepresent disclosure also provides kits containing the C1q antibodies ofthis disclosure (e.g., C1q neutralizing antibodies of this disclosure).

Complement Proteins

The antibodies of this disclosure specifically recognize complementfactor C1q and/or C1q in the C1 complex of the classical complementactivation pathway. The recognized complement factor may be derived,without limitation, from any organism having a complement system,including any mammalian organism such as human, mouse, rat, rabbit,monkey, dog, cat, cow, horse, camel, sheep, goat, or pig.

As used herein “C1 complex” refers to a protein complex that mayinclude, without limitation, one C1q protein, two C1r proteins, and twoC1s proteins (e.g., C1qr²s²).

As used herein “complement factor C1q” refers to both wild typesequences and naturally occurring variant sequences.

A non-limiting example of a complement factor C1q recognized byantibodies of this invention is human C1q, including the threepolypeptide chains A, B, and C:

C1q, chain A (homo sapiens), Accession No. Protein Data Base:NP_057075.1; GenBank No.: NM_015991:

>gi|7705753|ref|NP_057075.1| complement C1q subcomponent subunit Aprecursor [Homo sapiens]

(SEQ ID NO: 1) MEGPRGWLVLCVLAISLASMVTEDLCRAPDGKKGEAGRPGRRGRPGLKGEQGEPGAPGIRTGIQGLKGDQGEPGPSGNPGKVGYPGPSGPLGARGIPGIKGTKGSPGNIKDQPRPAFSAIRRNPPMGGNVVIFDTVITNQEEPYQNHSGRFVCTVPGYYYFTFQVLSQWEICLSIVSSSRGQVRRSLGFCDTTNKGLFQVVSGGMVLQLQQGDQVWVEKDPKKGHIYQGSEADSVFSGFLIFPSAC1q, chain B (homo sapiens), Accession No. Protein Data Base:NP_000482.3; GenBank No.: NM_000491.3:>gi|87298828|ref|NP_000482.3| complement C1q subcomponent subunit Bprecursor [Homo sapiens]

(SEQ ID NO: 2) MMMKIPWGSIPVLMLLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIKGEKGLPGLAGDHGEFGEKGDPGIPGNPGKVGPKGPMGPKGGPGAPGAPGPKGESGDYKATQKIAFSATRTINVPLRRDQTIRFDHVITNMNNNYEPRSGKFTCKVPGLYYFTYHASSRGNLCVNLMRGRERAQKVVTFCDYAYNTFQVTTGGMVLKLEQGENVFLQATDKNSLLGMEGANSIFSGFLLFPD MEA C1q, chain C (homo sapiens), Accession No. Protein Data Base:NP_001107573.1; GenBank No.: NM_001114101.1:>gi∥66235903|ref|NP_001107573.1| complement C1q subcomponent subunit Cprecursor [Homo sapiens]

(SEQ ID NO: 3) MDVGPSSLPHLGLKLLLLLLLLPLRGQANTGCYGIPGMPGLPGAPGKDGYDGLPGPKGEPGIPAIPGIRGPKGQKGEPGLPGHPGKNGPMGPPGMPGVPGPMGIPGEPGEEGRYKQKFQSVFTVTRQTHQPPAPNSLIRFNAVLTNPQGDYDTSTGKFTCKVPGLYYFVYHASHTANLCVLLYRSGVKVVTFCGHTSKTNQVNSGGVLLRLQVGEEVWLAVNDYYDMVGIQGSDSVFSGFLLFPD 

Accordingly, an anti-C1q antibody of the present disclosure may bind topolypeptide chain A, polypeptide chain B, and/or polypeptide chain C ofa C1q protein. In some embodiments, an anti-C1q antibody of the presentdisclosure binds to polypeptide chain A, polypeptide chain B, and/orpolypeptide chain C of human C1q or a homolog thereof, such as mouse,rat, rabbit, monkey, dog, cat, cow, horse, camel, sheep, goat, or pigC1q.

Anti-C1q Antibodies

The antibodies of this disclosure specifically bind to a complementfactor C1q and/or C1q in the C1 complex of the classical complementpathway. In some embodiments, the anti-C1q antibodies specifically bindto human C1q. In some embodiments, the anti-C1q antibodies specificallybind to human and mouse C1q. In some embodiments, the anti-C1qantibodies specifically bind to rat C1q. In some embodiments, theanti-C1q antibodies specifically bind to human C1q, mouse C1q, and ratC1q.

In some embodiments, the anti-C1q antibodies of this disclosureneutralize a biological activity of complement factor C1q. In someembodiments, the antibodies inhibit the interaction between complementfactor C1q and other complement factors, such as C1r or C1s or betweenC1q and an antibody, such as an autoantibody. As disclosed herein, anautoantibody of the present disclosure includes, without limitation, anantibody that recognizes a host antigen and activates the classicalpathway of complement activation. In the first step of this activationprocess complement factor C1q binds to theautoantibody-autoantigen-immune complex. In some embodiments, theantibodies inhibit the interaction between complement factor C1q and anon-complement factor. A non-complement factor may includephosphatidylserine, pentraxin-3, C-reactive protein (CRP), globular C1qreceptor (gC1qR), complement receptor 1 (CR1), (3-amyloid, andcalreticulin. In some embodiments, the antibodies inhibit the classicalcomplement activation pathway. In certain embodiments, the antibodiesfurther inhibit the alternative pathway. In some embodiments, theantibodies inhibit autoantibody- and complement-dependent cytotoxicity(CDC). In some embodiments, the antibodies inhibit complement-dependentcell-mediated cytotoxicity (CDCC). In some embodiments, the antibodiesinhibit B-cell antibody production, dendritic cell maturation, T-cellproliferation, cytokine production, or microglia activation. In someembodiments, the antibodies inhibit the Arthus reaction. In someembodiments, the antibodies inhibit phagocytosis of synapses or nerveendings. In some embodiments, the antibodies inhibit the activation ofcomplement receptor 3 (CR3/C3) expressing cells.

The functional properties of the antibodies of this invention, such asdissociation constants for antigens, inhibition of protein-proteininteractions (e.g., C1q-autoantibody interactions), inhibition ofautoantibody-dependent and complement-dependent cytotoxicity (CDC),inhibition of complement-dependent cell-mediated cytotoxicity (CDCC), orlesion formation, may, without limitation, be measured in in vitro, exvivo, or in vivo experiments.

The dissociation constants (K_(D)) of the anti-C1q antibodies for C1qmay be less than 100 nM, less than 90 nM, less than 80 nM, less than 70nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM,less than 20 nM, less than 10 nM, less than 9 nM, less than 8 nM, lessthan 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM,less than 0.05 nM, less than 0.01 nM, or less than 0.005 nM. In someembodiments, dissociation constants range from less than about 30 nM toless than about 100 pM. In some embodiments, dissociation constants areless than about 30 nM. In some embodiments, dissociation constants areless than about 20 nM. In some embodiments, dissociation constants areless than about 10 nM. In some embodiments, dissociation constants areless than about 5 nM. In some embodiments, dissociation constants areless than about 1 nM. In some embodiments, dissociation constants areless than about 100 pM. In certain embodiments, the dissociationconstants of the anti-C1q antibody range from less than about 30 nM toless than about 100 pM for human C1q, and range from less than about 30nM to less than about 100 pM for mouse C1q. In certain embodiments,dissociation constants of the anti-C1q antibody are less than about 30nM for human C1q and less than about 30 nM for mouse C1q. In certainembodiments, dissociation constants of the anti-C1q antibody are lessthan about 20 nM for human C1q and less than about 20 nM for mouse C1q.In certain embodiments, dissociation constants of the anti-C1q antibodyare less than about 10 nM for human C1q and less than about 10 nM formouse C1q. In certain embodiments, dissociation constants of theanti-C1q antibody are less than about 5 nM for human C1q and less thanabout 5 nM for mouse C1q. In certain embodiments, dissociation constantsof the anti-C1q antibody are less than about 1 nM for human C1q and lessthan about 1 nM mouse C1q. In certain embodiments, the dissociationconstants of the anti-C1q antibody are less than 100 pM for human C1qand less than 100 pM for mouse C1q. Antibody dissociation constants forantigens other than C1q may be least 5-fold, at least 10-fold, at least100-fold, at least 1,000-fold, at least 10,000-fold, or at least100,000-fold higher that the dissociation constants for C1q. Forexample, the dissociation constant of a C1q antibody of this disclosuremay be at least 1,000-fold higher for C1s than for C1q. Dissociationconstants may be determined through any analytical technique, includingany biochemical or biophysical technique such as ELISA, surface plasmonresonance (SPR), bio layer interferometry (see, e.g., Octet System byForteBio), isothermal titration calorimetry (ITC), differential scanningcalorimetry (DSC), circular dichroism (CD), stopped-flow analysis, andcolorimetric or fluorescent protein melting analyses. Dissociationconstants (K_(D)) of the anti-C1q antibodies for C1q may be determined,e.g., using full-length antibodies or antibody fragments, such as Fabfragments.

One exemplary way of determining binding affinity of antibodies to C1qis by measuring binding affinity of monofunctional Fab fragments of theantibody. To obtain monofunctional Fab fragments, an antibody (forexample, IgG) can be cleaved with papain or expressed recombinantly. Theaffinity of an Fab fragment of an antibody can be determined by surfaceplasmon resonance (Biacore3000™ surface plasmon resonance (SPR) system,Biacore™, INC, Piscataway N.J.) equipped with pre-immobilizedstreptavidin sensor chips (SA) using HBS-EP running buffer (0.01M HEPES,pH 7.4, 0.15 NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20). Biotinylatedhuman C1q (or any other C1q) can be diluted into HBS-EP buffer to aconcentration of less than 0.5 μg/mL and injected across the individualchip channels using variable contact times, to achieve two ranges ofantigen density, either 50-200 response units (RU) for detailed kineticstudies or 800-1,000 RU for screening assays. Regeneration studies haveshown that 25 mM NaOH in 25% v/v ethanol effectively removes the boundFab while keeping the activity of C1q on the chip for over 200injections. Typically, serial dilutions (spanning concentrations of0.1-10.times. estimated K_(D)) of purified Fab samples are injected for1 min at 100 μL/minute and dissociation times of up to 2 hours areallowed. The concentrations of the Fab proteins are determined by ELISAand/or SDS-PAGE electrophoresis using a Fab of known concentration (asdetermined by amino acid analysis) as a standard. Kinetic associationrates (k_(on)) and dissociation rates (k_(off)) are obtainedsimultaneously by fitting the data globally to a 1:1 Langmuir bindingmodel (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). MethodsEnzymology 6. 99-110) using the BIAevaluation program. Equilibriumdissociation constant (K_(D)) values are calculated as k_(off)/k_(on).This protocol is suitable for use in determining binding affinity of anantibody to any C1q, including human C1q, C1q of another mammal (such asmouse C1q, rat C1q, primate C1q), as well as different forms of C1q.Binding affinity of an antibody is generally measured at 25° C., but canalso be measured at 37° C.

The antibodies of this disclosure may bind to C1q antigens derived fromany organism having a complement system, including any mammalianorganism such as human, mouse, rat, rabbit, monkey, dog, cat, cow,horse, camel, sheep, goat, or pig. In some embodiments, the anti-C1qantibodies bind specifically to epitopes on human C1q. In someembodiments, the anti-C1q antibodies specifically bind to epitopes onboth human and mouse C1q. In some embodiments, the anti-C1q antibodiesspecifically bind to epitopes on human, mouse, and rat C1q.

In some embodiments, provided herein is an anti-C1q antibody that bindsto an epitope of C1q that is the same as or overlaps with the C1qepitope bound by another antibody of this disclosure. In certainembodiments, provided herein is an anti-C1q antibody that binds to anepitope of C1q that is the same as or overlaps with the C1q epitopebound by anti-C1q antibody M1. In some embodiments, the anti-C1qantibody competes with another antibody of this disclosure for bindingto C1q. In certain embodiments, the anti-C1q antibody competes withanti-C1q antibody M1 or an antigen-binding fragment thereof for bindingto C1q.

Methods that may be used to determine which C1q epitope of an anti-C1qantibody binds to, or whether two antibodies bind to the same or anoverlapping epitope, may include, without limitation, X-raycrystallography, NMR spectroscopy, Alanine-Scanning Mutagenesis, thescreening of peptide libraries that include C1q-derived peptides withoverlapping C1q sequences, and competition assays. Competition assaysare especially useful to determine whether two antibodies bind the sameepitope by recognizing identical or sterically overlapping epitopes orwhether one antibody competitively inhibits binding of another antibodyto the antigen. These assays are known in the art. Typically, an antigenor antigen expressing cells are immobilized on a multi-well plate andthe ability of unlabeled antibodies to block the binding of labeledantibodies is measured. Common labels for such competition assays areradioactive labels or enzyme labels.

Competitive antibodies encompassed herein are antibodies that inhibit(i.e., prevent or interfere with in comparison to a control) or reducethe binding of any anti-C1q antibody of this disclosure (such as M1 oran antigen-binding fragment of M1) to C1q by at least 50%, 60%, 70%,80%, 90% and 95% at 1 μM or less. For example, the concentrationcompeting antibody in the competition assay may be at or below the K_(D)of antibody M1 or an antigen-binding fragment of M1. Competition betweenbinding members may be readily assayed in vitro for example using ELISAand/or by monitoring the interaction of the antibodies with C1q insolution. The exact means for conducting the analysis is not critical.C1q may be immobilized to a 96-well plate or may be placed in ahomogenous solution. In specific embodiments, the ability of unlabeledcandidate antibody(ies) to block the binding of the labeled anti-C1qantibody, e.g. M1, can be measured using radioactive, enzyme or otherlabels. In the reverse assay, the ability of unlabeled antibodies tointerfere with the interaction of a labeled anti-C1q antibody with C1qwherein said labeled anti-C1q antibody, e.g., M1, and C1q are alreadybound is determined. The readout is through measurement of bound label.C1q and the candidate antibody(ies) may be added in any order or at thesame time.

In some embodiments, the anti-C1q antibody inhibits the interactionbetween C1q and an autoantibody. In some embodiments, the anti-C1qantibody is murine anti-human C1q monoclonal antibody M1, which isproduced by a hybridoma cell line deposited with ATCC on Jun. 6, 2013with ATCC Accession Number PTA-120399.

In some embodiments, the anti-C1q antibody is an isolated antibody whichbinds essentially the same C1q epitope as M1. In some embodiments, theanti-C1q antibody is an isolated antibody comprising the HVR-L1, HVR-L2,and HVR-L3 of the light chain variable domains of monoclonal antibody M1produced by the hybridoma cell line deposited with ATCC on Jun. 6, 2013with ATCC Accession Number PTA-120399, or progeny thereof. In someembodiments, the anti-C1q antibody is an isolated antibody comprisingthe HVR-H1, HVR-H2, and HVR-H3 of the heavy chain variable domains ofmonoclonal antibody M1 produced by the hybridoma cell line depositedwith ATCC on Jun. 6, 2013 with ATCC Accession Number PTA-120399, orprogeny thereof. In some embodiments, the anti-C1q antibody is anisolated antibody comprising the HVR-L1, HVR-L2, and HVR-L3 of the lightchain variable domains and the HVR-H1, HVR-H2, and HVR-H3 of the heavychain variable domains of monoclonal antibody M1 produced by thehybridoma cell line deposited with ATCC on Jun. 6, 2013 with ATCCAccession Number PTA-120399, or progeny thereof.

In some embodiments, the anti-C1q antibody binds to a C1q protein andbinds to one or more amino acids of the C1q protein within amino acidresidues selected from (a) amino acid residues 196-226 of SEQ ID NO:1(SEQ ID NO:16), or amino acid residues of a C1q protein chain A (C1qA)corresponding to amino acid residues 196-226(GLFQVVSGGMVLQLQQGDQVWVEKDPKKGHI) of SEQ ID NO:1 (SEQ ID NO:16); (b)amino acid residues 196-221 of SEQ ID NO:1 (SEQ ID NO:17), or amino acidresidues of a C1qA corresponding to amino acid residues 196-221(GLFQVVSGGMVLQLQQGDQVWVEKDP) of SEQ ID. NO:1 (SEQ ID NO:17); (c) aminoacid residues 202-221 of SEQ ID NO:1 (SEQ ID NO:18), or amino acidresidues of a C1qA corresponding to amino acid residues 202-221(SGGMVLQLQQGDQVWVEKDP) of SEQ ID NO:1 (SEQ ID NO:18); (d) amino acidresidues 202-219 of SEQ ID NO:1 (SEQ ID NO:19), or amino acid residuesof a C1qA corresponding to amino acid residues 202-219(SGGMVLQLQQGDQVWVEK) of SEQ ID NO:1 (SEQ ID NO:19); and (e) amino acidresidues Lys 219 and/or Ser 202 of SEQ ID NO:1, or amino acid residuesof a C1qA corresponding Lys 219 and/or Ser 202 of SEQ ID NO:1.

In some embodiments, the antibody further binds to one or more aminoacids of the C1q protein within amino acid residues selected from: (a)amino acid residues 218-240 of SEQ ID NO:3 (SEQ ID NO:20) or amino acidresidues of a C1q protein chain C (C1qC) corresponding to amino acidresidues 218-240 (WLAVNDYYDMVGI QGSDSVFSGF) of SEQ ID NO:3 (SEQ IDNO:20); (b) amino acid residues 225-240 of SEQ ID NO:3 (SEQ ID NO:21) oramino acid residues of a C1qC corresponding to amino acid residues225-240 (YDMVGI QGSDSVFSGF) of SEQ ID NO:3 (SEQ ID NO:21); (c) aminoacid residues 225-232 of SEQ ID NO:3 (SEQ ID NO:22) or amino acidresidues of a C1qC corresponding to amino acid residues 225-232(YDMVGIQG) of SEQ ID NO:3 (SEQ ID NO:22); (d) amino acid residue Tyr 225of SEQ ID NO:3 or an amino acid residue of a C1qC corresponding to aminoacid residue Tyr 225 of SEQ ID NO:3; (e) amino acid residues 174-196 ofSEQ ID NO:3 (SEQ ID NO:23) or amino acid residues of a C1qCcorresponding to amino acid residues 174-196 (HTANLCVLLYRSGVKVVTFCGHT)of SEQ ID NO:3 (SEQ ID NO:23); (f) amino acid residues 184-192 of SEQ IDNO:3 (SEQ ID NO:24) or amino acid residues of a C1qC corresponding toamino acid residues 184-192 (RSGVKVVTF) of SEQ ID NO:3 (SEQ ID NO:24);(g) amino acid residues 185-187 of SEQ ID NO:3 or amino acid residues ofa C1qC corresponding to amino acid residues 185-187 (SGV) of SEQ IDNO:3; (h) amino acid residue Ser 185 of SEQ ID NO:3 or an amino acidresidue of a C1qC corresponding to amino acid residue Ser 185 of SEQ IDNO:3.

In certain embodiments, the anti-C1q antibody binds to amino acidresidue Lys 219 and Ser 202 of the human C1qA as shown in SEQ ID NO:1 oramino acids of a human C1qA corresponding to Lys 219 and Ser 202 asshown in SEQ ID NO:1, and amino acid residue Tyr 225 of the human C1qCas shown in SEQ ID NO:3 or an amino acid residue of a human C1qCcorresponding to Tyr 225 as shown in SEQ ID NO:3. In certainembodiments, the anti-C1q antibody binds to amino acid residue Lys 219of the human C1qA as shown in SEQ ID NO:1 or an amino acid residue of ahuman C1qA corresponding to Lys 219 as shown in SEQ ID NO:1, and aminoacid residue Ser 185 of the human C1qC as shown in SEQ ID NO:3 or anamino acid residue of a human C1qC corresponding to Ser 185 as shown inSEQ ID NO:3.

In some embodiments, the anti-C1q antibody binds to a C1q protein andbinds to one or more amino acids of the C1q protein within amino acidresidues selected from: (a) amino acid residues 218-240 of SEQ ID NO:3(SEQ ID NO:20) or amino acid residues of a C1qC corresponding to aminoacid residues 218-240 (WLAVNDYYDMVGI QGSDSVFSGF) of SEQ ID NO:3 (SEQ IDNO:20); (b) amino acid residues 225-240 of SEQ ID NO:3 (SEQ ID NO:21) oramino acid residues of a C1qC corresponding to amino acid residues225-240 (YDMVGI QGSDSVFSGF) of SEQ ID NO:3 (SEQ ID NO:21); (c) aminoacid residues 225-232 of SEQ ID NO:3 (SEQ ID NO:22) or amino acidresidues of a C1qC corresponding to amino acid residues 225-232(YDMVGIQG) of SEQ ID NO:3 (SEQ ID NO:22); (d) amino acid residue Tyr 225of SEQ ID NO:3 or an amino acid residue of a C1qC corresponding to aminoacid residue Tyr 225 of SEQ ID NO:3; (e) amino acid residues 174-196 ofSEQ ID NO:3 (SEQ ID NO:23) or amino acid residues of a C1qCcorresponding to amino acid residues 174-196 (HTANLCVLLYRSGVKVVTFCGHT)of SEQ ID NO:3 (SEQ ID NO:23); (f) amino acid residues 184-192 of SEQ IDNO:3 (SEQ ID NO:24) or amino acid residues of a C1qC corresponding toamino acid residues 184-192 (RSGVKVVTF) of SEQ ID NO:3 (SEQ ID NO:24);(g) amino acid residues 185-187 of SEQ ID NO:3 or amino acid residues ofa C1qC corresponding to amino acid residues 185-187 (SGV) of SEQ IDNO:3; (h) amino acid residue Ser 185 of SEQ ID NO:3 or an amino acidresidue of a C1qC corresponding to amino acid residue Ser 185 of SEQ IDNO:3.

In some embodiments, the anti-C1q antibody of this disclosure inhibitsthe interaction between C1q and C1s. In some embodiments, the anti-C1qantibody inhibits the interaction between C1q and C1r. In someembodiments the anti-C1q antibody inhibits the interaction between C1qand Cis and between C1q and C1r. In some embodiments, the anti-C1qantibody inhibits the interaction between C1q and another antibody, suchas an autoantibody. In some embodiments, the anti-C1q antibody inhibitsthe respective interactions, at a stoichiometry of less than 2.5:1;2.0:1; 1.5:1; or 1.0:1. In some embodiments, the C1q antibody inhibitsan interaction, such as the C1q-C1s interaction, at approximatelyequimolar concentrations of C1q and the anti-C1q antibody. In otherembodiments, the anti-C1q antibody binds to C1q with a stoichiometry ofless than 20:1; less than 19.5:1; less than 19:1; less than 18.5:1; lessthan 18:1; less than 17.5:1; less than 17:1; less than 16.5:1; less than16:1; less than 15.5:1; less than 15:1; less than 14.5:1; less than14:1; less than 13.5:1; less than 13:1; less than 12.5:1; less than12:1; less than 11.5:1; less than 11:1; less than 10.5:1; less than10:1; less than 9.5:1; less than 9:1; less than 8.5:1; less than 8:1;less than 7.5:1; less than 7:1; less than 6.5:1; less than 6:1; lessthan 5.5:1; less than 5:1; less than 4.5:1; less than 4:1; less than3.5:1; less than 3:1; less than 2.5:1; less than 2.0:1; less than 1.5:1;or less than 1.0:1. In certain embodiments, the anti-C1q antibody bindsC1q with a binding stoichiometry that ranges from 20:1 to 1.0:1 or lessthan 1.0:1. In certain embodiments, the anti-C1q antibody binds C1q witha binding stoichiometry that ranges from 6:1 to 1.0:1 or less than1.0:1. In certain embodiments, the anti-C1q antibody binds C1q with abinding stoichiometry that ranges from 2.5:1 to 1.0:1 or less than1.0:1. In some embodiments, the anti-C1q antibody inhibits theinteraction between C1q and C1r, or between C1q and C1s, or between C1qand both C1r and C1s. In some embodiments, the anti-C1q antibodyinhibits the interaction between C1q and C1r, between C1q and C1s,and/or between C1q and both C1r and C1s. In some embodiments, theanti-C1q antibody binds to the C1q A-chain. In other embodiments, theanti-C1q antibody binds to the C1q B-chain. In other embodiments, theanti-C1q antibody binds to the C1q C-chain. In some embodiments, theanti-C1q antibody binds to the C1q A-chain, the C1q B-chain and/or theC1q C-chain. In some embodiments, the anti-C1q antibody binds to theglobular domain of the C1q A-chain, B-chain, and/or C-chain. In otherembodiments, the anti-C1q antibody binds to the collagen-like domain ofthe C1q A-chain, the C1q B-chain, and/or the C1q C-chain.

Where antibodies of this disclosure inhibit the interaction between twoor more complement factors, such as the interaction of C1q and C1s, orthe interaction between C1q and C1r, the interaction occurring in thepresence of the antibody may be reduced by at least 10%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or at least 99% relative to acontrol wherein the antibodies of this disclosure are absent. In certainembodiments, the interaction occurring in the presence of the antibodyis reduced by an amount that ranges from at least 30% to at least 99%relative to a control wherein the antibodies of this disclosure areabsent.

In some embodiments, the antibodies of this disclosure inhibitC4-cleavage by at least 20%, at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, orat least 99%, or by an amount that ranges from at least 30% to at least99%, relative to a control wherein the antibodies of this disclosure areabsent. Methods for measuring C4-cleavage are well known in the art. TheEC₅₀ values for antibodies of this disclosure with respect C4-cleavagemay be less than 3 μg/ml; 2.5 μg/ml; 2.0 μg/ml; 1.5 μg/ml; 1.0 μg/ml;0.5 μg/ml; 0.25 μg/ml; 0.1 μg/ml; 0.05 μg/ml. In some embodiments, theantibodies of this disclosure inhibit C4-cleavage at approximatelyequimolar concentrations of C1q and the respective anti-C1q antibody.

In some embodiments, the antibodies of this disclosure inhibitautoantibody-dependent and complement-dependent cytotoxicity (CDC) by atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, or at least 99%, orby an amount that ranges from at least 30% to at least 99%, relative toa control wherein the antibodies of this disclosure are absent. The EC₅₀values for antibodies of this disclosure with respect to inhibition ofautoantibody-dependent and complement-dependent cytotoxicity may be lessthan 3 μg/ml; 2.5 μg/ml; 2.0 μg/ml; 1.5 μg/ml; 1.0 μg/ml; 0.5 μg/ml;0.25 μg/ml; 0.1 μg/ml; 0.05 μg/ml.

In some embodiments, the antibodies of this disclosure inhibitcomplement-dependent cell-mediated cytotoxicity (CDCC) by at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or at least 99%, or by an amountthat ranges from at least 30% to at least 99%, relative to a controlwherein the antibodies of this disclosure are absent. Methods formeasuring CDCC are well known in the art. The EC₅₀ values for antibodiesof this disclosure with respect CDCC inhibition may be 1 less than 3μg/ml; 2.5 μg/ml; 2.0 μg/ml; 1.5 μg/ml; 1.0 μg/ml; 0.5 μg/ml; 0.25μg/ml; 0.1 μg/ml; 0.05 μg/ml. In some embodiments, the antibodies ofthis disclosure inhibit CDCC but not antibody-dependent cellularcytotoxicity (ADCC).

In some embodiments, the antibodies of this disclosure inhibit C1Fhemolysis (also referred to as CH50 hemolysis) by at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, or at least 99%, or by an amount thatranges from at least 30% to at least 99%, relative to a control whereinthe antibodies of this disclosure are absent or wherein controlantibodies are used that do not bind to a complement factor or anotherantibody such as an autoantibody (see, e.g., Example 3). Methods formeasuring C1F hemolysis are well known in the art (see, e.g., Example3). The EC₅₀ values for antibodies of this disclosure with respect toC1F hemolysis may be less than 3 μg/ml; 2.5 μg/ml; 2.0 μg/ml; 1.5 μg/ml;1.0 μg/ml; 0.5 μg/ml; 0.25 μg/ml; 0.1 μg/ml; 0.05 μg/ml. In someembodiments, the anti-C1q antibodies of this disclosure neutralize atleast 50% of C1F hemolysis at a dose of less than 200 ng/ml, less than100 ng/ml, less than 50 ng/ml, or less than 20 ng/ml. In someembodiments, the antibodies of this disclosure neutralize C1F hemolysisat approximately equimolar concentrations of C1q and the anti-C1qantibody. In some embodiments, the anti-C1q antibodies of thisdisclosure neutralize hemolysis in a human C1F hemolysis assay. In someembodiments, the antibodies of this disclosure neutralize hemolysis in ahuman, mouse, and rat C1F hemolysis assay (see, e.g., Example 3).

In some embodiments, the alternative pathway may amplify CDC initiatedby C1q binding and subsequent C1s activation; in at least some of theseembodiments, the antibodies of this disclosure inhibit the alternativepathway by at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, or at least 99%, orby an amount that ranges from at least 30% to at least 99%, relative toa control wherein the antibodies of this disclosure were absent.

In some embodiments, the antibodies of this disclosure prevent synapticloss in a cellular in vitro model or an in vivo model of synaptic loss,such as an in vivo mouse model. In vivo mouse models may include Tg2576,a mouse amyloid precursor protein (APP) transgenic model of Alzheimer'sdisease, R6/2 NT-CAG150, a transgenic model for Huntington's disease, orSMAΔ7, a mouse model for Spinal Muscular Atrophy, or DBA/2J, a geneticmouse model of glaucoma. In general, any neurodegenerative disease modelmay be used that displays synapse loss.

Methods for measuring synaptic loss in vitro or in vivo are well knownin the art. In vitro lesion formation may be reduced by at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or at least 95%, or by an amount that ranges from at least30% to at least 95%, relative to a control experiment in whichantibodies of this disclosure are absent. The EC₅₀ values for antibodiesof this disclosure with respect to the prevention of in vitro lesionformation may be less than 3 μg/ml; 2.5 μg/ml; 2.0 μg/ml; 1.5 μg/ml; 1.0μg/ml; 0.5 μg/ml; 0.25 μg/ml; 0.1 μg/ml; 0.05 μg/ml. In vivo synapticloss may be reduced by at least 5%, at least 10%, at least 15%, at least20%, at least 35%, at least 40%, or at least 50%, or by an amount thatranges from at least 5% to at least 50%, relative to a controlexperiment in which antibodies of this disclosure are absent.

In some embodiments, the antibodies of this disclosure prevent lesionformation in an ex vivo spinal cord slice model of NMO or in an in vivomouse model of NMO. Methods for measuring lesion formation ex vivo or invivo are well known in the art. Ex vivo lesion formation may be reducedat least by a relative score of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, or4.0. The EC₅₀ values for antibodies of this disclosure with respect tothe prevention of ex vivo lesion formation may be less than 3 μg/ml;less than 2.5 μg/ml; less than 2.0 μg/ml; less than 1.5 μg/ml; less than1.0 μg/ml; less than 0.5 μg/ml; less than 0.25 μg/ml; less than 0.1μg/ml; or less than 0.05 μg/ml. In vivo lesion formation may be reducedby at least 5%, at least 10%, at least 15%, at least 20%, at least 35%,at least 40%, or at least 50%, or by an amount that ranges from at least5% to at least 50%, in terms of loss of staining (% of area). Stainingmay be assessed, without limitation, by APQ4 staining, GFAP staining, orMBP staining.

The present disclosure provides anti-C1q antibodies. The antibodies ofthis disclosure may have one or more of the following characteristics.The antibodies of this disclosure may be polyclonal antibodies,monoclonal antibodies, humanized antibodies, human antibodies, antibodyfragments, bispecific and polyspecific antibodies, multivalentantibodies, or heteroconjugate antibodies. Antibody fragments of thisdisclosure may be functional fragments that bind the same epitope as anyof the anti-C1q antibodies of this disclosure. In some embodiments, theantibody fragments of this disclosure specifically bind to andneutralize a biological activity of C1q. In some embodiments, theantibody fragments are miniaturized versions of the anti-C1q antibodiesor antibody fragments of this disclosure that have the same epitope ofthe corresponding full-length antibody, but have much smaller moleculeweight. Such miniaturized anti-C1q antibody fragments may have betterbrain penetration ability and a shorter half-life, which is advantageousfor imaging and diagnostic utilities (see e.g., Lütje S et al.,Bioconjug Chem. 2014 Feb. 19; 25(2):335-41; Tavaré R et al., Proc NatlAcad Sci USA. 2014 Jan. 21; 111(3):1108-13; and Wiehr S et al.,Prostate. 2014 May; 74(7):743-55). Accordingly, in some embodiments,anti-C1q antibody fragments of this disclosure have better brainpenetration as compared to their corresponding full-length antibodiesand/or have a shorter half-life as compared to their correspondingfull-length antibodies. In some embodiments, anti-C1q antibodies of thepresent disclosure are bispecific antibodies recognizing a first antigenand a second antigen. In some embodiments, the first antigen is a C1qantigen. In some embodiments, the second antigen is an antigenfacilitating transport across the blood-brain-barrier, including withoutlimitation, transferrin receptor (TR), insulin receptor (HIR),insulin-like growth factor receptor (IGFR), low-density lipoproteinreceptor related proteins 1 and 2 (LPR-1 and 2), diphtheria toxinreceptor, CRM197, a llama single domain antibody, TMEM 30(A), a proteintransduction domain, TAT, Syn-B, penetratin, a poly-arginine peptide, anangiopep peptide, and ANG1005. The antibodies of this disclosure mayfurther contain engineered effector functions, amino acid sequencemodifications or other antibody modifications known in the art; e.g.,the constant region of the anti-C1q antibodies described herein may bemodified to impair complement activation.

Additional anti-C1q antibodies, e.g., antibodies that specifically bindto a C1q protein of the present disclosure, may be identified, screened,and/or characterized for their physical/chemical properties and/orbiological activities by various assays known in the art.

Antibody Preparation

Anti-C1q antibodies of the present disclosure can encompass polyclonalantibodies, monoclonal antibodies, humanized antibodies, chimericantibodies, human antibodies, antibody fragments (e.g., Fab, Fab′-SH,Fv, scFv, and F(ab′)₂ fragments), bispecific and polyspecificantibodies, multivalent antibodies, heteroconjugate antibodies, libraryderived antibodies, antibodies having modified effector functions,fusion proteins containing an antibody portion, and any other modifiedconfiguration of the immunoglobulin molecule that includes an antigenrecognition site, such as an epitope having amino acid residues of a C1qprotein of the present disclosure, including glycosylation variants ofantibodies, amino acid sequence variants of antibodies, and covalentlymodified antibodies. The anti-C1q antibodies may be human, murine, rat,or of any other origin (including chimeric or humanized antibodies).

(1) Polyclonal Antibodies

Polyclonal antibodies, such as polyclonal anti-C1q antibodies, aregenerally raised in animals by multiple subcutaneous (sc) orintraperitoneal (ip) injections of the relevant antigen and an adjuvant.It may be useful to conjugate the relevant antigen (e.g., a purified orrecombinant C1q protein of the present disclosure) to a protein that isimmunogenic in the species to be immunized, e.g., keyhole limpethemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybeantrypsin inhibitor, using a bifunctional or derivatizing agent, e.g.,maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, where R and R¹are independently lower alkyl groups. Examples of adjuvants which may beemployed include Freund's complete adjuvant and MPL-TDM adjuvant(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). Theimmunization protocol may be selected by one skilled in the art withoutundue experimentation.

The animals are immunized against the desired antigen, immunogenicconjugates, or derivatives by combining, e.g., 100 μg (for rabbits) or 5μg (for mice) of the protein or conjugate with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later, the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to fourteen days later,the animals are bled and the serum is assayed for antibody titer.Animals are boosted until the titer plateaus. Conjugates also can bemade in recombinant-cell culture as protein fusions. Also, aggregatingagents such as alum are suitable to enhance the immune response.

(2) Monoclonal Antibodies

Monoclonal antibodies, such as monoclonal anti-C1q antibodies, areobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations and/orpost-translational modifications (e.g., isomerizations, amidations) thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies.

For example, the monoclonal anti-C1q antibodies may be made using thehybridoma method first described by Kohler et al., Nature, 256:495(1975), or may be made by recombinant DNA methods (U.S. Pat. No.4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization (e.g., apurified or recombinant C1q protein of the present disclosure).Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The immunizing agent will typically include the antigenic protein (e.g.,a purified or recombinant C1q protein of the present disclosure) or afusion variant thereof. Generally peripheral blood lymphocytes (“PBLs”)are used if cells of human origin are desired, while spleen or lymphnode cells are used if non-human mammalian sources are desired. Thelymphoctyes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell. Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress (1986), pp. 59-103.

Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine or human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells thusprepared are seeded and grown in a suitable culture medium that maycontain one or more substances that inhibit the growth or survival ofthe unfused, parental myeloma cells. For example, if the parentalmyeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (HATmedium), which are substances that prevent the growth ofHGPRT-deficient-cells.

In some embodiments, immortalized myeloma cells are those that fuseefficiently, support stable high-level production of antibody by theselected antibody-producing cells, and are sensitive to a medium such asHAT medium. Among these, are murine myeloma lines, such as those derivedfrom MOPC-21 and MPC-11 mouse tumors (available from the Salk InstituteCell Distribution Center, San Diego, Calif. USA), as well as SP-2 cellsand derivatives thereof (e.g., X63-Ag8-653) (available from the AmericanType Culture Collection, Manassas, Va. USA). Human myeloma andmouse-human heteromyeloma cell lines have also been described for theproduction of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen (e.g.,a C1q protein of the present disclosure). In some embodiments, thebinding specificity of monoclonal antibodies produced by hybridoma cellsis determined by immunoprecipitation or by an in vitro binding assay,such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay(ELISA).

The culture medium in which the hybridoma cells are cultured can beassayed for the presence of monoclonal antibodies directed against thedesired antigen (e.g., a C1q protein of the present disclosure). In someembodiments, the binding affinity and specificity of the monoclonalantibody can be determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linked assay(ELISA). Such techniques and assays are known in the in art. Forexample, binding affinity may be determined by the Scatchard analysis ofMunson et al., Anal. Biochem., 107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as tumors in a mammal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose chromatography, hydroxylapatitechromatography, gel electrophoresis, dialysis, affinity chromatography,and other methods as described above.

Anti-C1q monoclonal antibodies may also be made by recombinant DNAmethods, such as those disclosed in U.S. Pat. No. 4,816,567, and asdescribed above. DNA encoding the monoclonal antibodies is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that specifically bind to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells serveas a source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host-cells such asE. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, inorder to synthesize monoclonal antibodies in such recombinanthost-cells. Review articles on recombinant expression in bacteria of DNAencoding the antibody include Skerra et al., Curr. Opin. Immunol.,5:256-262 (1993) and Plückthun, Immunol. Rev. 130:151-188 (1992).

In certain embodiments, anti-C1q antibodies can be isolated fromantibody phage libraries generated using the techniques described inMcCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991)described the isolation of murine and human antibodies, respectively,from phage libraries. Subsequent publications describe the production ofhigh affinity (nanomolar (“nM”) range) human antibodies by chainshuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), as well ascombinatorial infection and in vivo recombination as a strategy forconstructing very large phage libraries (Waterhouse et al., Nucl. AcidsRes., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies of desired specificity (e.g., thosethat bind a C1q protein of the present disclosure).

The DNA encoding antibodies or fragments thereof may also be modified,for example, by substituting the coding sequence for human heavy- andlight-chain constant domains in place of the homologous murine sequences(U.S. Pat. No. 4,816,567; Morrison, et al., Proc. Natl Acad. Sci. USA,81:6851 (1984)), or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. Typically such non-immunoglobulin polypeptides aresubstituted for the constant domains of an antibody, or they aresubstituted for the variable domains of one antigen-combining site of anantibody to create a chimeric bivalent antibody comprising oneantigen-combining site having specificity for an antigen and anotherantigen-combining site having specificity for a different antigen.

The monoclonal antibodies described herein (e.g., anti-C1q antibodies ofthe present disclosure or fragments thereof) may by monovalent, thepreparation of which is well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain and amodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues may be substituted withanother amino acid residue or are deleted so as to prevent crosslinking.In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart.

Chimeric or hybrid anti-C1q antibodies also may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide-exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate.

(3) Humanized Antibodies

Anti-C1q antibodies of the present disclosure or antibody fragmentsthereof may further include humanized or human antibodies. Humanizedforms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such asFab, Fab′-SH, Fv, scFv, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. Humanized antibodies include human immunoglobulins(recipient antibody) in which residues from a complementaritydetermining region (CDR) of the recipient are replaced by residues froma CDR of a non-human species (donor antibody) such as mouse, rat orrabbit having the desired specificity, affinity and capacity. In someinstances, Fv framework residues of the human immunoglobulin arereplaced by corresponding non-human residues. Humanized antibodies mayalso comprise residues which are found neither in the recipient antibodynor in the imported CDR or framework sequences. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally willalso comprise at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin. Jones et al., Nature321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988) andPresta, Curr. Opin. Struct. Biol. 2: 593-596 (1992). In someembodiments, the anti-C1q antibody is a chimeric antibody comprising theheavy and light chain variable domains of any of the anti-C1q antibodydescribed herein (e.g., antibody M1 and 4A4B11) and constant regionsfrom a human immunoglobulin.

Methods for humanizing non-human anti-C1q antibodies are well known inthe art. Generally, a humanized antibody has one or more amino acidresidues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers, Jones et al., Nature 321:522-525 (1986); Riechmann etal., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536(1988), or through substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody. Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies. Carter et al., Proc. Nat'l Acad. Sci.USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993).

Furthermore, it is important that antibodies be humanized with retentionof high affinity for the antigen and other favorable biologicalproperties. To achieve this goal, humanized antibodies are prepared by aprocess of analyzing the parental sequences and various conceptualhumanized products using three-dimensional models of the parental andhumanized sequences. Three-dimensional immunoglobulin models arecommonly available and are familiar to those skilled in the art.Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the recipient andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen or antigens (e.g., C1qproteins of the present disclosure), is achieved. In general, the CDRresidues are directly and most substantially involved in influencingantigen binding.

Various forms of the humanized anti-C1q antibody are contemplated. Forexample, the humanized anti-C1q antibody may be an antibody fragment,such as an Fab, which is optionally conjugated with one or morecytotoxic agent(s) in order to generate an immunoconjugate.Alternatively, the humanized anti-C1q antibody may be an intactantibody, such as an intact IgG1 antibody.

(4) Human Antibodies

Alternatively, human anti-C1q antibodies can be generated. For example,it is now possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Thehomozygous deletion of the antibody heavy-chain joining region (JO genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. Transfer of the human germ-lineimmunoglobulin gene array in such germ-line mutant mice will result inthe production of human antibodies upon antigen challenge. See, e.g.,Jakobovits et al., Proc. Nat'l Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Yearin Immunol., 7:33 (1993); U.S. Pat. No. 5,591,669 and WO 97/17852.

Alternatively, phage display technology can be used to produce humananti-C1q antibodies and antibody fragments in vitro, from immunoglobulinvariable (V) domain gene repertoires from unimmunized donors. McCaffertyet al., Nature 348:552-553 (1990); Hoogenboom and Winter, J. Mol. Biol.227: 381 (1991). According to this technique, antibody V domain genesare cloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, such as M13 or fd, and displayed asfunctional antibody fragments on the surface of the phage particle.Because the filamentous particle contains a single-stranded DNA copy ofthe phage genome, selections based on the functional properties of theantibody also result in selection of the gene encoding the antibodyexhibiting those properties. Thus, the phage mimics some of theproperties of the B-cell. Phage display can be performed in a variety offormats, reviewed in, e.g., Johnson, Kevin S. and Chiswell, David J.,Curr. Opin Struct. Biol. 3:564-571 (1993). Several sources of V-genesegments can be used for phage display. Clackson et al., Nature352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodiesfrom a small random combinatorial library of V genes derived from thespleens of immunized mice. A repertoire of V genes from unimmunizedhuman donors can be constructed and antibodies to a diverse array ofantigens (including self-antigens) can be isolated essentially followingthe techniques described by Marks et al., J. Mol. Biol. 222:581-597(1991), or Griffith et al., EMBO J. 12:725-734 (1993). See also U.S.Pat. Nos. 5,565,332 and 5,573,905. Additionally, yeast displaytechnology can be used to produce human anti-C1q antibodies and antibodyfragments in vitro (e.g., WO 2009/036379; WO 2010/105256; WO2012/009568; US 2009/0181855; US 2010/0056386; and Feldhaus and Siegel(2004) J. Immunological Methods 290:69-80). In other embodiments,ribosome display technology can be used to produce human anti-C1qantibodies and antibody fragments in vitro (e.g., Roberts and Szostak(1997) Proc Natl Acad Sci 94:12297-12302; Schaffitzel et al. (1999) J.Immunolical Methods 231:119-135; Lipovsek and Plückthun (2004) J.Immunological Methods 290:51-67).

The techniques of Cole et al., and Boerner et al., are also availablefor the preparation of human anti-C1q monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol. 147(1): 86-95 (1991). Similarly,human anti-C1q antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806,5,569,825, 5,625,126, 5,633,425, 5,661,016 and in the followingscientific publications: Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature368: 812-13 (1994), Fishwild et al., Nature Biotechnology 14: 845-51(1996), Neuberger, Nature Biotechnology 14: 826 (1996) and Lonberg andHuszar, Intern. Rev. Immunol. 13: 65-93 (1995).

Finally, human anti-C1q antibodies may also be generated in vitro byactivated B-cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

(5) Antibody Fragments

In certain embodiments there are advantages to using anti-C1q antibodyfragments, rather than whole anti-C1q antibodies. Smaller fragment sizesallow for rapid clearance.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., J. Biochem.Biophys. Method. 24:107-117 (1992); and Brennan et al., Science 229:81(1985)). However, these fragments can now be produced directly byrecombinant host-cells, for example, using nucleic acids encodinganti-C1q antibodies of the present disclosure. Fab, Fv and scFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the straightforward production of large amounts of thesefragments. A anti-C1q antibody fragments can also be isolated from theantibody phage libraries as discussed above. Alternatively, Fab′-SHfragments can be directly recovered from E. coli and chemically coupledto form F(ab′)₂ fragments (Carter et al., Bio/Technology 10:163-167(1992)). According to another approach, F(ab′)₂ fragments can beisolated directly from recombinant host-cell culture. Production of Faband F(ab′)₂ antibody fragments with increased in vivo half-lives aredescribed in U.S. Pat. No. 5,869,046. In other embodiments, the antibodyof choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S.Pat. No. 5,571,894 and U.S. Pat. No. 5,587,458. The anti-C1q, anti-C1r,or anti-C1q antibody fragment may also be a “linear antibody,” e.g., asdescribed in U.S. Pat. No. 5,641,870. Such linear antibody fragments maybe monospecific or bispecific.

(6) Bispecific and Polyspecific Antibodies

Bispecific antibodies (BsAbs) are antibodies that have bindingspecificities for at least two different epitopes, including those onthe same or another protein (e.g., one or more C1q proteins of thepresent disclosure). Alternatively, one part of a BsAb can be armed tobind to the target C1q antigen, and another can be combined with an armthat binds to a second protein. Such antibodies can be derived from fulllength antibodies or antibody fragments (e.g., F(ab′)₂ bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy-chain/light chain pairs,where the two chains have different specificities. Millstein et al.,Nature, 305:537-539 (1983). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion may bewith an immunoglobulin heavy chain constant domain, comprising at leastpart of the hinge, C_(H)2, and C_(H)3 regions. In some embodiments, thefirst heavy-chain constant region (C_(H)1) containing the site necessaryfor light chain binding is present in at least one of the fusions. DNAsencoding the immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Thisprovides for great flexibility in adjusting the mutual proportions ofthe three polypeptide fragments in embodiments when unequal ratios ofthe three polypeptide chains used in the construction provide theoptimum yields. It is, however, possible to insert the coding sequencesfor two or all three polypeptide chains in one expression vector whenthe expression of at least two polypeptide chains in equal ratiosresults in high yields or when the ratios are of no particularsignificance.

In some embodiments of this approach, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyhalf of the bispecific molecules provides for an easy way of separation.This approach is disclosed in WO 94/04690. For further details ofgenerating bispecific antibodies, see, for example, Suresh et al.,Methods in Enzymology 121: 210 (1986).

According to another approach described in WO 96/27011 or U.S. Pat. No.5,731,168, the interface between a pair of antibody molecules can beengineered to maximize the percentage of heterodimers which arerecovered from recombinant-cell culture. The interface may comprise atleast a part of the C_(H)3 region of an antibody constant domain. Inthis method, one or more small amino acid side chains from the interfaceof the first antibody molecule are replaced with larger side chains(e.g., tyrosine or tryptophan). Compensatory “cavities” of identical orsimilar size to the large side chains(s) are created on the interface ofthe second antibody molecule by replacing large amino acid side chainswith smaller ones (e.g., alanine or threonine). This provides amechanism for increasing the yield of the heterodimer over otherunwanted end-products such as homodimers.

Techniques for generating bispecific antibodies from antibody fragmentshave been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science 229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-TNB derivative to form the bispecificantibody. The bispecific antibodies produced can be used as agents forthe selective immobilization of enzymes.

Fab′ fragments may be directly recovered from E. coli and chemicallycoupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describes the production of fully humanized bispecificantibody F(ab′)₂ molecules. Each Fab′ fragment was separately secretedfrom E. coli and subjected to directed chemical coupling in vitro toform the bispecific antibody. The bispecific antibody thus formed wasable to bind to cells overexpressing the ErbB2 receptor and normal humanT-cells, as well as trigger the lytic activity of human cytotoxiclymphocytes against human breast tumor targets.

Various techniques for making and isolating bivalent antibody fragmentsdirectly from recombinant-cell culture have also been described. Forexample, bivalent heterodimers have been produced using leucine zippers.Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucinezipper peptides from the Fos and Jun proteins were linked to the Fab′portions of two different antibodies by gene fusion. The antibodyhomodimers were reduced at the hinge region to form monomers and thenre-oxidized to form the antibody heterodimers. The “diabody” technologydescribed by Hollinger et al., Proc. Nat'l Acad. Sci. USA, 90: 6444-6448(1993) has provided an alternative mechanism for makingbispecific/bivalent antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and VH domains of another fragment, thereby forming two antigen-bindingsites. Another strategy for making bispecific/bivalent antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are also contemplated. Forexample, trispecific antibodies can be prepared. Tutt et al., J.Immunol. 147:60 (1991).

Exemplary bispecific antibodies may bind to two different antigens. Insome embodiments a bispecific antibody binds to a first antigen, C1q,and a second antigen facilitating transport across the blood-brainbarrier. Numerous antigens are known in the art that facilitatetransport across the blood-brain barrier (see, e.g., Gabathuler R.,Approaches to transport therapeutic drugs across the blood-brain barrierto treat brain diseases, Neurobiol. Dis. 37 (2010) 48-57). Such secondantigens include, without limitation, transferrin receptor (TR), insulinreceptor (HIR), Insulin-like growth factor receptor (IGFR), low-densitylipoprotein receptor related proteins 1 and 2 (LPR-1 and 2), diphtheriatoxin receptor, including CRM197 (a non-toxic mutant of diphtheriatoxin), llama single domain antibodies such as TMEM 30(A) (Flippase),protein transduction domains such as TAT, Syn-B, or penetratin,poly-arginine or generally positively charged peptides, and Angiopeppeptides such as ANG1005 (see, e.g., Gabathuler, 2010).

(7) Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The anti-C1q antibodies of the present disclosure orantibody fragments thereof can be multivalent antibodies (which areother than of the IgM class) with three or more antigen binding sites(e.g., tetravalent antibodies), which can be readily produced byrecombinant expression of nucleic acid encoding the polypeptide chainsof the antibody. The multivalent antibody can comprise a dimerizationdomain and three or more antigen binding sites. In some embodiments, thedimerization domain comprises an Fc region or a hinge region. In thisscenario, the antibody will comprise an Fc region and three or moreantigen binding sites amino-terminal to the Fc region. In someembodiments, the multivalent antibody herein contains three to abouteight, and in some embodiments four, antigen binding sites. Themultivalent antibody contains at least one polypeptide chain (and insome embodiments two polypeptide chains), wherein the polypeptide chainor chains comprise two or more variable domains. For instance, thepolypeptide chain or chains may comprise VD1-(X1)n-VD2-(X2)n-Fc, whereinVD1 is a first variable domain, VD2 is a second variable domain, Fc isone polypeptide chain of an Fc region, X1 and X2 represent an amino acidor polypeptide, and n is 0 or 1. Similarly, the polypeptide chain orchains may comprise V_(H)-C_(H)1-flexible linker-V_(H)-C_(H)1-Fc regionchain; or V_(H)-C_(H)1-V_(H)-C_(H)1-Fc region chain. The multivalentantibody herein may further comprise at least two (and in someembodiments four) light chain variable domain polypeptides. Themultivalent antibody herein may, for instance, comprise from about twoto about eight light chain variable domain polypeptides. The light chainvariable domain polypeptides contemplated here comprise a light chainvariable domain and, optionally, further comprise a CL domain.

(8) Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentdisclosure. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies (e.g., anti-C1q antibodies of the present disclosureor antibody fragments thereof). For example, one of the antibodies inthe heteroconjugate can be coupled to avidin, the other to biotin. Suchantibodies have, for example, been proposed to target immune systemcells to unwanted cells, U.S. Pat. No. 4,676,980, and have been used totreat HIV infection. International Publication Nos. WO 91/00360, WO92/200373 and EP 0308936. It is contemplated that the antibodies may beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinsmay be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980. Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

(9) Effector Function Engineering

It may also be desirable to modify an anti-C1q antibody of the presentdisclosure to modify effector function and/or to increase serumhalf-life of the antibody. For example, the Fc receptor binding site onthe constant region may be modified or mutated to remove or reducebinding affinity to certain Fc receptors, such as FcγRI, FcγRII, and/orFcγRIII. In some embodiments, the effector function is impaired byremoving N-glycosylation of the Fc region (e.g., in the CH 2 domain ofIgG) of the antibody. In some embodiments, the effector function isimpaired by modifying regions such as 233-236, 297, and/or 327-331 ofhuman IgG as described in PCT WO 99/58572 and Armour et al., MolecularImmunology 40: 585-593 (2003); Reddy et al., J. Immunology 164:1925-1933(2000).

The constant region of the anti-complement antibodies described hereinmay also be modified to impair complement activation. For example,complement activation of IgG antibodies following binding of the C1component of complement may be reduced by mutating amino acid residuesin the constant region in a C1 binding motif (e.g., C1q binding motif).It has been reported that Ala mutation for each of D270, K322, P329,P331 of human IgG1 significantly reduced the ability of the antibody tobind to C1q and activating complement. For murine IgG2b, C1q bindingmotif constitutes residues E318, K320, and K322. Idusogie et al. (2000)J. Immunology 164:4178-4184; Duncan et al. (1988) Nature 322: 738-740.As the C1s binding motif E318, K320, and K322 identified for murineIgG2b is believed to be common for other antibody isotypes (Duncan etal. (1988) Nature 322:738-740), C1q binding activity for IgG2b can beabolished by replacing any one of the three specified residues with aresidue having an inappropriate functionality on its side chain. It isnot necessary to replace the ionic residues only with Ala to abolish C1qbinding. It is also possible to use other alkyl-substituted non-ionicresidues, such as Gly, Ile, Leu, or Val, or such aromatic non-polarresidues as Phe, Tyr, Trp and Pro in place of any one of the threeresidues in order to abolish C1q binding. In addition, it is alsopossible to use such polar non-ionic residues as Ser, Thr, Cys, and Metin place of residues 320 and 322, but not 318, in order to abolish C1sbinding activity. In addition, removal of carbohydrate modifications ofthe Fc region necessary for complement binding can prevent complementactivation Glycosylation of a conserved asparagine (Asn-297) on the CH2domain of IgG heavy chains is essential for antibody effector functions(Jefferis et al. (1998) Immunol Rev 163:59-76). Modification of the Fcglycan alters IgG conformation and reduces the Fc affinity for bindingof complement protein C1q and effector cell receptor FcR (Alhorn et al.(2008) PLos ONE 2008; 3:e1413). Complete removal of the Fc glycanabolishes CDC and ADCC. Deglycosylation can be performed usingglycosidase enzymes for example Endoglycosidase S (EndoS), a 108 kDaenzyme encoded by the gene endoS of Streptococcus pyogenes thatselectively digests asparagine-linked glycans on the heavy chain of allIgG subclasses, without action on other immunoglobulin classes or otherglycoproteins (Collin et al. (2001) EMBO J 2001; 20:3046-3055).

To increase the serum half-life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

(10) Other Amino Acid Sequence Modifications

Amino acid sequence modifications of anti-C1q antibodies of the presentdisclosure, or antibody fragments thereof, are also contemplated. Forexample, it may be desirable to improve the binding affinity and/orother biological properties of the antibodies or antibody fragments.Amino acid sequence variants of the antibodies or antibody fragments areprepared by introducing appropriate nucleotide changes into the nucleicacid encoding the antibodies or antibody fragments, or by peptidesynthesis. Such modifications include, for example, deletions from,and/or insertions into and/or substitutions of, residues within theamino acid sequences of the antibody. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics(i.e., the ability to bind or physically interact with a C1q protein ofthe present disclosure). The amino acid changes also may alterpost-translational processes of the antibody, such as changing thenumber or position of glycosylation sites.

A useful method for identification of certain residues or regions of theanti-C1q antibody that are preferred locations for mutagenesis is called“alanine scanning mutagenesis” as described by Cunningham and Wells inScience, 244:1081-1085 (1989). Here, a residue or group of targetresidues are identified (e.g., charged residues such as arg, asp, his,lys, and glu) and replaced by a neutral or negatively charged amino acid(most preferably alanine or polyalanine) to affect the interaction ofthe amino acids with the target antigen. Those amino acid locationsdemonstrating functional sensitivity to the substitutions then arerefined by introducing further or other variants at, or for, the sitesof substitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, alanine scanning or random mutagenesis isconducted at the target codon or region and the expressed antibodyvariants are screened for the desired activity.

Amino acid sequence insertions include amino- (“N”) and/or carboxy-(“C”) terminal fusions ranging in length from one residue topolypeptides containing a hundred or more residues, as well asintrasequence insertions of single or multiple amino acid residues.Examples of terminal insertions include an antibody with an N-terminalmethionyl residue or the antibody fused to a cytotoxic polypeptide.Other insertional variants of the antibody molecule include the fusionto the N- or C-terminus of the antibody to an enzyme or a polypeptidewhich increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative substitutions are shownin the Table A below under the heading of “preferred substitutions”. Ifsuch substitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table A,or as further described below in reference to amino acid classes, may beintroduced and the products screened.

TABLE A Amino Acid Substitutions Original Exemplary Preferred Residuesubstitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln;asn lys Asn (N) gln; his; asp, lys; arg gln Asp (D) glu; asn glu Cys (C)ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; gln asp Gly (G) ala alaHis (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe;norleucine leu Leu (L) norleucine; ile; val; met; ala; phe ile Lys (K)arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala;tyr tyr Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phetyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala;norleucine leu

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability (particularly where the antibody is an antibodyfragment, such as an Fv fragment).

In some embodiments, the substitutional variant involves substitutingone or more hypervariable region residues of a parent antibody (e.g. ahumanized or human anti-C1q antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g., 6-7 sites) are mutated togenerate all possible amino substitutions at each site. The antibodyvariants thus generated are displayed in a monovalent fashion fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g., binding affinity) asherein disclosed. In order to identify candidate hypervariable regionsites for modification, alanine scanning mutagenesis can be performed toidentify hypervariable region residues contributing significantly toantigen binding. Alternatively, or additionally, it may be beneficial toanalyze a crystal structure of the antigen-antibody complex to identifycontact points between the antibody and the antigen (e.g., a C1q proteinof the present disclosure). Such contact residues and neighboringresidues are candidates for substitution according to the techniqueselaborated herein. Once such variants are generated, the panel ofvariants is subjected to screening as described herein and antibodieswith superior properties in one or more relevant assays may be selectedfor further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of theanti-IgE antibody are prepared by a variety of methods known in the art.These methods include, but are not limited to, isolation from a naturalsource (in the case of naturally occurring amino acid sequence variants)or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of the antibodies (e.g.,anti-C1q antibody of the present disclosure) or antibody fragments.

(11) Other Antibody Modifications

Anti-C1q antibodies of the present disclosure, or antibody fragmentsthereof, can be further modified to contain additional non-proteinaceousmoieties that are known in the art and readily available. In someembodiments, the moieties suitable for derivatization of the antibodyare water-soluble polymers. Non-limiting examples of water-solublepolymers include, but are not limited to, polyethylene glycol (PEG),copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose,dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, polypropylene glycol homopolymers,polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols(e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethyleneglycol propionaldehyde may have advantages in manufacturing due to itsstability in water. The polymer may be of any molecular weight, and maybe branched or unbranched. The number of polymers attached to theantibody may vary, and if more than one polymer is attached, they can bethe same or different molecules. In general, the number and/or type ofpolymers used for derivatization can be determined based onconsiderations including, but not limited to, the particular propertiesor functions of the antibody to be improved, whether the antibodyderivative will be used in a therapy under defined conditions, etc. Suchtechniques and other suitable formulations are disclosed in Remington:The Science and Practice of Pharmacy, 20th Ed., Alfonso Gennaro, Ed.,Philadelphia College of Pharmacy and Science (2000).

Nucleic Acids, Vectors, and Host Cells

Anti-C1q antibodies of the present disclosure may be produced usingrecombinant methods and compositions, e.g., as described in U.S. Pat.No. 4,816,567. In some embodiments, isolated nucleic acids having anucleotide sequence encoding any of the anti-C1q antibodies of thepresent disclosure are provided. Such nucleic acids may encode an aminoacid sequence containing the VL and/or an amino acid sequence containingthe VH of the anti-C1q antibody (e.g., the light and/or heavy chains ofthe antibody). In some embodiments, one or more vectors (e.g.,expression vectors) containing such nucleic acids are provided. In someembodiments, a host cell containing such nucleic acid is also provided.In some embodiments, the host cell contains (e.g., has been transducedwith): (1) a vector containing a nucleic acid that encodes an amino acidsequence containing the VL of the antibody and an amino acid sequencecontaining the VH of the antibody, or (2) a first vector containing anucleic acid that encodes an amino acid sequence containing the VL ofthe antibody and a second vector containing a nucleic acid that encodesan amino acid sequence containing the VH of the antibody. In someembodiments, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary(CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell).

Methods of making an anti-C1q antibody of the present disclosure areprovided. In some embodiments, the method includes culturing a host cellof the present disclosure containing a nucleic acid encoding theanti-C1q antibody, under conditions suitable for expression of theantibody. In some embodiments, the antibody is subsequently recoveredfrom the host cell (or host cell culture medium). See also Example 1.

For recombinant production of an anti-C1q antibody of the presentdisclosure, a nucleic acid encoding the anti-C1q antibody is isolatedand inserted into one or more vectors for further cloning and/orexpression in a host cell. Such nucleic acid may be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of the antibody).

Suitable vectors containing a nucleic acid sequence encoding any of theanti-C1q antibodies of the present disclosure, or fragments thereofpolypeptides (including antibodies) described herein include, withoutlimitation, cloning vectors and expression vectors. Suitable cloningvectors can be constructed according to standard techniques, or may beselected from a large number of cloning vectors available in the art.While the cloning vector selected may vary according to the host cellintended to be used, useful cloning vectors generally have the abilityto self-replicate, may possess a single target for a particularrestriction endonuclease, and/or may carry genes for a marker that canbe used in selecting clones containing the vector. Suitable examplesinclude plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript(e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1,pCR1, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. Theseand many other cloning vectors are available from commercial vendorssuch as BioRad, Strategene, and Invitrogen.

Expression vectors generally are replicable polynucleotide constructsthat contain a nucleic acid of the present disclosure. The expressionvector may replicable in the host cells either as episomes or as anintegral part of the chromosomal DNA. Suitable expression vectorsinclude but are not limited to plasmids, viral vectors, includingadenoviruses, adeno-associated viruses, retroviruses, cosmids, andexpression vector(s) disclosed in PCT Publication No. WO 87/04462.Vector components may generally include, but are not limited to, one ormore of the following: a signal sequence; an origin of replication; oneor more marker genes; suitable transcriptional controlling elements(such as promoters, enhancers and terminator). For expression (i.e.,translation), one or more translational controlling elements are alsousually required, such as ribosome binding sites, translation initiationsites, and stop codons.

The vectors containing the nucleic acids of interest can be introducedinto the host cell by any of a number of appropriate means, includingelectroporation, transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (e.g., where thevector is an infectious agent such as vaccinia virus). The choice ofintroducing vectors or polynucleotides will often depend on features ofthe host cell. In some embodiments, the vector contains a nucleic acidcontaining one or more amino acid sequences encoding an anti-C1qantibody of the present disclosure.

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells. For example, anti-C1qantibodies of the present disclosure may be produced in bacteria, inparticular when glycosylation and Fc effector function are not needed.For expression of antibody fragments and polypeptides in bacteria (e.g.,U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523; and Charlton,Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press,Totowa, N.J., 2003), pp. 245-254, describing expression of antibodyfragments in E. coli.). After expression, the antibody may be isolatedfrom the bacterial cell paste in a soluble fraction and can be furtherpurified.

In addition to prokaryotes, eukaryotic microorganisms, such asfilamentous fungi or yeast, are also suitable cloning or expressionhosts for antibody-encoding vectors, including fungi and yeast strainswhose glycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern (e.g., Gerngross, Nat. Biotech. 22:1409-1414 (2004); and Li etal., Nat. Biotech. 24:210-215 (2006)).

Suitable host cells for the expression of glycosylated antibody can alsobe derived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells. Plant cell cultures can also be utilized ashosts (e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978,and 6,417,429, describing PLANTIBODIES™ technology for producingantibodies in transgenic plants.).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

Pharmaceutical Compositions

Anti-C1q antibodies of the present disclosure can be incorporated into avariety of formulations for therapeutic use (e.g., by administration) orin the manufacture of a medicament (e.g., for treating or preventing aneurodegenerative disease or autoimmune disease) by combining theantibodies with appropriate pharmaceutically acceptable carriers ordiluents, and may be formulated into preparations in solid, semi-solid,liquid or gaseous forms. Examples of such formulations include, withoutlimitation, tablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants, gels, microspheres, and aerosols.Pharmaceutical compositions can include, depending on the formulationdesired, pharmaceutically-acceptable, non-toxic carriers of diluents,which are vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents include, without limitation, distilled water, bufferedwater, physiological saline, PBS, Ringer's solution, dextrose solution,and Hank's solution. A pharmaceutical composition or formulation of thepresent disclosure can further include other carriers, adjuvants, ornon-toxic, nontherapeutic, nonimmunogenic stabilizers, excipients andthe like. The compositions can also include additional substances toapproximate physiological conditions, such as pH adjusting and bufferingagents, toxicity adjusting agents, wetting agents and detergents.

A pharmaceutical composition of the present disclosure can also includeany of a variety of stabilizing agents, such as an antioxidant forexample. When the pharmaceutical composition includes a polypeptide, thepolypeptide can be complexed with various well-known compounds thatenhance the in vivo stability of the polypeptide, or otherwise enhanceits pharmacological properties (e.g., increase the half-life of thepolypeptide, reduce its toxicity, and enhance solubility or uptake).Examples of such modifications or complexing agents include, withoutlimitation, sulfate, gluconate, citrate and phosphate. The polypeptidesof a composition can also be complexed with molecules that enhance theirin vivo attributes. Such molecules include, without limitation,carbohydrates, polyamines, amino acids, other peptides, ions (e.g.,sodium, potassium, calcium, magnesium, manganese), and lipids.

Further examples of formulations that are suitable for various types ofadministration can be found in Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, Pa., 17th ed. (1985). For a briefreview of methods for drug delivery, see, Langer, Science 249:1527-1533(1990).

For oral administration, the active ingredient can be administered insolid dosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. The activecomponent(s) can be encapsulated in gelatin capsules together withinactive ingredients and powdered carriers, such as glucose, lactose,sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesiumstearate, stearic acid, sodium saccharin, talcum, magnesium carbonate.Examples of additional inactive ingredients that may be added to providedesirable color, taste, stability, buffering capacity, dispersion orother known desirable features are red iron oxide, silica gel, sodiumlauryl sulfate, titanium dioxide, and edible white ink. Similar diluentscan be used to make compressed tablets. Both tablets and capsules can bemanufactured as sustained release products to provide for continuousrelease of medication over a period of hours. Compressed tablets can besugar coated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric-coated for selectivedisintegration in the gastrointestinal tract. Liquid dosage forms fororal administration can contain coloring and flavoring to increasepatient acceptance.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.

The components used to formulate the pharmaceutical compositions arepreferably of high purity and are substantially free of potentiallyharmful contaminants (e.g., at least National Food (NF) grade, generallyat least analytical grade, and more typically at least pharmaceuticalgrade). Moreover, compositions intended for in vivo use are usuallysterile. To the extent that a given compound must be synthesized priorto use, the resulting product is typically substantially free of anypotentially toxic agents, particularly any endotoxins, which may bepresent during the synthesis or purification process. Compositions forparental administration are also sterile, substantially isotonic andmade under GMP conditions.

Formulations may be optimized for retention and stabilization in thebrain or central nervous system. When the agent is administered into thecranial compartment, it is desirable for the agent to be retained in thecompartment, and not to diffuse or otherwise cross the blood brainbarrier. Stabilization techniques include cross-linking, multimerizing,or linking to groups such as polyethylene glycol, polyacrylamide,neutral protein carriers, etc. in order to achieve an increase inmolecular weight.

Other strategies for increasing retention include the entrapment of theantibody, such as an anti-C1q antibody of the present disclosure, in abiodegradable or bioerodible implant. The rate of release of thetherapeutically active agent is controlled by the rate of transportthrough the polymeric matrix, and the biodegradation of the implant. Thetransport of drug through the polymer barrier will also be affected bycompound solubility, polymer hydrophilicity, extent of polymercross-linking, expansion of the polymer upon water absorption so as tomake the polymer barrier more permeable to the drug, geometry of theimplant, and the like. The implants are of dimensions commensurate withthe size and shape of the region selected as the site of implantation.Implants may be particles, sheets, patches, plaques, fibers,microcapsules and the like and may be of any size or shape compatiblewith the selected site of insertion.

The implants may be monolithic, i.e. having the active agenthomogenously distributed through the polymeric matrix, or encapsulated,where a reservoir of active agent is encapsulated by the polymericmatrix. The selection of the polymeric composition to be employed willvary with the site of administration, the desired period of treatment,patient tolerance, the nature of the disease to be treated and the like.Characteristics of the polymers will include biodegradability at thesite of implantation, compatibility with the agent of interest, ease ofencapsulation, a half-life in the physiological environment.

Biodegradable polymeric compositions which may be employed may beorganic esters or ethers, which when degraded result in physiologicallyacceptable degradation products, including the monomers. Anhydrides,amides, orthoesters or the like, by themselves or in combination withother monomers, may find use. The polymers will be condensationpolymers. The polymers may be cross-linked or non-cross-linked. Ofparticular interest are polymers of hydroxyaliphatic carboxylic acids,either homo- or copolymers, and polysaccharides. Included among thepolyesters of interest are polymers of D-lactic acid, L-lactic acid,racemic lactic acid, glycolic acid, polycaprolactone, and combinationsthereof. By employing the L-lactate or D-lactate, a slowly biodegradingpolymer is achieved, while degradation is substantially enhanced withthe racemate. Copolymers of glycolic and lactic acid are of particularinterest, where the rate of biodegradation is controlled by the ratio ofglycolic to lactic acid. The most rapidly degraded copolymer has roughlyequal amounts of glycolic and lactic acid, where either homopolymer ismore resistant to degradation. The ratio of glycolic acid to lactic acidwill also affect the brittleness of in the implant, where a moreflexible implant is desirable for larger geometries. Among thepolysaccharides of interest are calcium alginate, and functionalizedcelluloses, particularly carboxymethylcellulose esters characterized bybeing water insoluble, a molecular weight of about 5 kD to 500 kD, etc.Biodegradable hydrogels may also be employed in the implants of thesubject invention. Hydrogels are typically a copolymer material,characterized by the ability to imbibe a liquid. Exemplary biodegradablehydrogels which may be employed are described in Heller in: Hydrogels inMedicine and Pharmacy, N. A. Peppes ed., Vol. III, CRC Press, BocaRaton, Fla., 1987, pp 137-149.

Pharmaceutical Dosages

Pharmaceutical compositions of the present disclosure containing ananti-C1q antibody of the present disclosure may be used (e.g.,administered to an individual in need of treatment with anti-C1qantibody, such as a human individual) in accord with known methods, suchas intravenous administration as a bolus or by continuous infusion overa period of time, by intramuscular, intraperitoneal, intracerobrospinal,intracranial, intraspinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes.

Dosages and desired drug concentration of pharmaceutical compositions ofthe present disclosure may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary artisan. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles described in Mordenti, J. andChappell, W. “The Use of Interspecies Scaling in Toxicokinetics,” InToxicokinetics and New Drug Development, Yacobi et al., Eds, PergamonPress, New York 1989, pp. 42-46.

For in vivo administration of any of the anti-C1q antibodies of thepresent disclosure, normal dosage amounts may vary from about 10 ng/kgup to about 100 mg/kg of an individual's body weight or more per day,depending upon the route of administration. In some embodiments, thedose amount is about 1 mg/kg/day to 10 mg/kg/day. For repeatedadministrations over several days or longer, depending on the severityof the disease, disorder, or condition to be treated, the treatment issustained until a desired suppression of symptoms is achieved.

An exemplary dosing regimen may include administering an initial dose ofan anti-C1q antibody, of about 2 mg/kg, followed by a weekly maintenancedose of about 1 mg/kg every other week. Other dosage regimens may beuseful, depending on the pattern of pharmacokinetic decay that thephysician wishes to achieve. For example, dosing an individual from oneto twenty-one times a week is contemplated herein. In certainembodiments, dosing ranging from about 3 μg/kg to about 2 mg/kg (such asabout 3 μg/kg, about 10 μg/kg, about 30 μg/kg, about 100 μg/kg, about300 μg/kg, about 1 mg/kg, or about 2 mg/kg) may be used. In certainembodiments, dosing frequency is three times per day, twice per day,once per day, once every other day, once weekly, once every two weeks,once every four weeks, once every five weeks, once every six weeks, onceevery seven weeks, once every eight weeks, once every nine weeks, onceevery ten weeks, or once monthly, once every two months, once everythree months, or longer. Progress of the therapy is easily monitored byconventional techniques and assays. The dosing regimen, including theanti-C1q antibody administered, can vary over time independently of thedose used.

Dosages for a particular anti-C1q antibody may be determined empiricallyin individuals who have been given one or more administrations of theanti-C1q antibody. Individuals are given incremental doses of ananti-C1q antibody. To assess efficacy of an anti-C1q antibody, anyclinical symptom of a neurodegenerative disorder, inflammatory disorder,or autoimmune disorder can be monitored.

Administration of an anti-C1q antibody of the present disclosure can becontinuous or intermittent, depending, for example, on the recipient'sphysiological condition, whether the purpose of the administration istherapeutic or prophylactic, and other factors known to skilledpractitioners. The administration of an anti-C1q antibody may beessentially continuous over a preselected period of time or may be in aseries of spaced doses.

Guidance regarding particular dosages and methods of delivery isprovided in the literature; see, for example, U.S. Pat. Nos. 4,657,760;5,206,344; or 5,225,212. It is within the scope of the invention thatdifferent formulations will be effective for different treatments anddifferent disorders, and that administration intended to treat aspecific organ or tissue may necessitate delivery in a manner differentfrom that to another organ or tissue. Moreover, dosages may beadministered by one or more separate administrations, or by continuousinfusion. For repeated administrations over several days or longer,depending on the condition, the treatment is sustained until a desiredsuppression of disease symptoms occurs. However, other dosage regimensmay be useful. The progress of this therapy is easily monitored byconventional techniques and assays.

Therapeutic Uses

The present disclosure provides anti-C1q antibodies, and antigen-bindingfragments thereof, which can bind to and neutralize a biologic activityof C1q. These anti-C1q antibodies are useful for preventing, reducingrisk, or treating a range of diseases associated with complementactivation, including, without limitation, neurodegenerative disorders,inflammatory disorders, and autoimmune disorders. Accordingly, asdisclosed herein, anti-C1q antibodies of the present disclosure may beused for treating, preventing, or reducing risk of a disease associatedwith complement activation, including, without limitation,neurodegenerative disorders, inflammatory disorders, and autoimmunedisorders, in an individual. In some embodiments, the individual hassuch a disease. In some embodiments, the individual is a human.

Neurodegenerative disorders that may be treated with anti-C1q antibodiesof this disclosure include disorders associated with loss of nerveconnections or synapses, including CF1-dependent synapse loss. Suchdisorders may include, without limitation, Alzheimer's disease,amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonicdystrophy, Down syndrome, Parkinson's disease, and Huntington's disease.In some neurodegenerative disorders, synapse loss is dependent on thecomplement receptor 3 (CR3)/C3 or complement receptor CR1. In someneurodegenerative disorders, synapse loss is associated withpathological activity-dependent synaptic pruning. In some disorders,synapses are phagocytosed by microglia. Accordingly, the anti-C1qantibodies of the present disclosure may be used to treat, prevent, orimprove one or more symptoms of a neurodegenerative disorder of thepresent disclosure. In some embodiments, the present disclosure providesmethods of treating, preventing, or improving one or more symptoms inindividuals having a neurodegenerative disorder of the presentdisclosure by administering an anti-C1q antibody of the presentdisclosure to, for example, inhibit the interaction between C1q and anautoantibody, such as an anti-ganglioside autoantibody, the interactionof C1q and C1r, and/or the interaction of C1q and C1s.

Inflammatory or autoimmune diseases that may be treated with anti-C1qantibodies of this disclosure include, without limitation, rheumatoidarthritis (RA), acute respiratory distress syndrome (ARDS), remotetissue injury after ischemia and reperfusion, complement activationduring cardiopulmonary bypass surgery, dermatomyositis, pemphigus, lupusnephritis and resultant glomerulonephritis and vasculitis,cardiopulmonary bypass, cardioplegia-induced coronary endothelialdysfunction, type II membranoproliferative glomerulonephritis, IgAnephropathy, acute renal failure, cryoglobulemia, antiphospholipidsyndrome, macular degenerative diseases, such as age-related maculardegeneration (AMD), choroidal neovascularization (CNV), uveitis,diabetic and other ischemia-related retinopathies, endophthalmitis, andother intraocular neovascular diseases, such as diabetic macular edema,pathological myopia, von Hippel-Lindau disease, histoplasmosis of theeye, Neuromyelitis Optica (NMO), Central Retinal Vein Occlusion (CRVO),corneal neovascularization, retinal neovascularization, as well asallo-transplantation, hyperacute rejection, hemodialysis, chronicocclusive pulmonary distress syndrome (COPD), asthma, and aspirationpneumonia. In some embodiments, autoimmune disease may further include,without limitation, Guillain-Barré syndrome, myasthenia gravis, Diabetesmellitus type 1, Hashimoto's thyroiditis, Addison's disease, Coeliacdisease, Crohn's disease, pernicious anaemia, Pemphigus vulgaris,vitiligo, autoimmune hemolytic anemias, paraneoplastic syndromes, avasculitis disease, polymyalgia rheumatica, temporal arteritis, andWegener's granulomatosis.

In autoimmune diseases, such as Neuromyelitis Optica (NMO),autoantibodies activate the complement system. In NMO patients, theclassical complement pathway is triggered by the binding of anautoantibody, such as an AQP4-targeted autoantibody, to its autoantigen,AQP4. AQP4 thereby activates the classical pathway of complementactivation. In the first step of this activation process complementfactor C1q binds to the autoantibody-autoantigen-immune complex.Autoantibodies may include naturally occurring antibodies, such as serumantibodies from NMO patients (commonly referred to as NMO-IgG) ormonoclonal antibodies, such as rAb-53.

Accordingly, the anti-C1q antibodies of the present disclosure may beused to treat, prevent, or improve one or more symptoms of aninflammatory or autoimmune disease of the present disclosure. In someembodiments, the present disclosure provides methods of treating,preventing, or improving one or more symptoms in individuals having aninflammatory or autoimmune disease of the present disclosure byadministering an anti-C1q antibody of the present disclosure to, forexample, inhibit the interaction between C1q and an autoantibody, suchas an anti-ganglioside autoantibody, the interaction of C1q and C1r,and/or the interaction of C1q and C1s.

Metabolic diseases that may be treated with anti-C1q antibodies include,without limitation, diabetes, such as type II diabetes, and obesity. Invitro and in vivo models of metabolic disorders that can be used for thetesting of anti-C1q antibodies are well known in the art. Accordingly,the anti-C1q antibodies of the present disclosure may be used to treat,prevent, or improve one or more symptoms of a metabolic disease of thepresent disclosure. In some embodiments, the present disclosure providesmethods of treating, preventing, or improving one or more symptoms inindividuals having metabolic disease of the present disclosure byadministering an anti-C1q antibody of the present disclosure to, forexample, inhibit the interaction between C1q and an autoantibody, suchas an anti-ganglioside autoantibody, the interaction of C1q and C1r,and/or the interaction of C1q and C1s.

Combination Treatments

The antibodies of the present disclosure may be used, withoutlimitation, in combination with any additional treatment forneurodegenerative disorders, inflammatory disorders, and/or autoimmunedisorders.

In some embodiments, an anti-C1q antibody of this disclosure isadministered in therapeutically effective amounts in combination with asecond anti-complement factor antibody (e.g., a neutralizinganti-complement factor antibody), such as an anti-C1s or anti-C1rantibody, or a second anti-C1q antibody. In some embodiments, ananti-C1q antibody of this disclosure is administered in therapeuticallyeffective amounts with a second and a third neutralizing anti-complementfactor antibody, such as a second anti-C1q antibody, an anti-C1santibody, and/or an anti-C1r antibody.

In some embodiments, the anti-C1q antibodies of this disclosure areadministered in combination with an inhibitor of antibody-dependentcellular cytotoxicity (ADCC). ADCC inhibitors may include, withoutlimitation, soluble NK cell inhibitory receptors such as the killer cellIg-like receptors (KIRs), which recognize HLA-A, HLA-B, or HLA-C andC-type lectin CD94/NKG2A heterodimers, which recognize HLA-E (see, e.g.,López-Botet M., T. Bellón, M. Llano, F. Navarro, P. Garcia & M. deMiguel. (2000), Paired inhibitory and triggering NK cell receptors forHLA class I molecules. Hum. Immunol. 61: 7-17; Lanier L. L. (1998)Follow the leader: NK cell receptors for classical and nonclassical MHCclass I. Cell 92: 705-707.), and cadmium (see, e.g., Immunopharmacology1990; Volume 20, Pages 73-8).

In some embodiments, the antibodies of this disclosure are administeredin combination with an inhibitor of the alternative pathway ofcomplement activation. Such inhibitors may include, without limitation,factor B blocking antibodies, factor D blocking antibodies, soluble,membrane-bound, tagged or fusion-protein forms of CD59, DAF, CR1, CR2,Crry or Comstatin-like peptides that block the cleavage of C3,non-peptide C3aR antagonists such as SB 290157, Cobra venom factor ornon-specific complement inhibitors such as nafamostat mesilate (FUTHAN;FUT-175), aprotinin, K-76 monocarboxylic acid (MX-1) and heparin (see,e.g., T. E. Mollnes & M. Kirschfink, Molecular Immunology 43 (2006)107-121). In some embodiments, the antibodies of this disclosure areadministered in combination with an inhibitor of the interaction betweenthe autoantibody and its autoantigen.

Such inhibitors may include purified soluble forms of the autoantigen,or antigen mimetics such as peptide or RNA-derived mimotopes, includingmimotopes of the AQP4 antigen. Alternatively, such inhibitors mayinclude blocking agents that recognize the autoantigen and preventbinding of the autoantibody without triggering the classical complementpathway. Such blocking agents may include, e.g., autoantigen-binding RNAaptamers or antibodies lacking functional C1q binding sites in their Fcdomains (e.g., Fab fragments or antibody otherwise engineered not tobind C1q).

Diagnostic Uses

The antibodies of this disclosure, or functional fragments thereof, alsohave diagnostic utility. This disclosure therefore provides for methodsof using the antibodies of this disclosure, or functional fragmentsthereof, for diagnostic purposes, such as the detection of C1q in anindividual or in tissue samples derived from an individual. In someembodiments, the individual is a human. In some embodiments, theindividual is a human patient suffering from a neurodegenerativedisorder or an inflammatory, or autoimmune disease. In some embodiments,the anti-C1q antibodies of this disclosure are used to detect synapsesand synapse loss. For example, synapse loss may be measured in anindividual suffering from a neurodegenerative disorder such asAlzheimer's disease or glaucoma.

In some embodiments, the diagnostic methods involve the steps ofadministering an anti-C1q antibody of this disclosure, or functionalfragment thereof, to an individual and detecting the antibody bound to asynapse of the individual. Antibody-binding to synapses may bequantified, for example, by non-invasive techniques such as positronemission tomography (PET), X-ray computed tomography, single-photonemission computed tomography (SPECT), computed tomography (CT), andcomputed axial tomography (CAT).

In some embodiments, the diagnostic methods involve detecting synapsesin a biological sample, such as a biopsy specimen, a tissue, or a cell.An anti-C1q antibody, or functional fragment thereof, is contacted withthe biological sample and synapse-bound antibody is detected. Thedetection method may involve quantification of the synapse-boundantibody. Antibody detection in biological samples may occur with anymethod known in the art, including immunofluorescence microscopy,immunocytochemistry, immunohistochemistry, ELISA, FACS analysis orimmunoprecipitation.

The quantification of synapse-bound antibodies provides a relativemeasure for the number of synapses present in the individual. Typically,synapses are quantified repeatedly over a period of time. The exactperiodicity of synapse quantification depends on many factors, includingthe nature of the neurodegenerative disease, the stage of diseaseprogression, treatment modalities and many other factors. Repeatmeasurements commonly reveal progressive synapse loss in individualshaving a neurodegenerative disorder. Alternatively, relative synapsecounts may be compared in populations of diseased individuals andhealthy control individuals at a single time point. In diseasedindividuals undergoing treatment, the treatment's efficacy can beassessed by comparing the rates of synapse loss in the treatedindividuals with the rates of synapse loss in a control group. Controlgroup members have received either no treatment or a control treatment,such as a placebo control.

Kits

The invention also provides kits containing an antibody of thisdisclosure, or a functional fragment thereof. Kits of the inventioninclude one or more containers comprising a purified anti-C1q antibodyof this disclosure. In some embodiments, the kits further includeinstructions for use in accordance with the methods of this disclosure.In some embodiments, these instructions comprise a description ofadministration of the anti-C1q antibody to treat or diagnose a diseaseassociated with complement activation including, without limitation aneurodegenerative disorder (e.g., Alzheimer's disease), inflammatorydisease, autoimmune disease, and/or metabolic disorder, according to anymethods of this disclosure. In some embodiments, the instructionscomprise a description of how to detect C1q, for example in anindividual, in a tissue sample, or in a cell. The kit may furthercomprise a description of selecting an individual suitable for treatmentbased on identifying whether that individual has the disease and thestage of the disease.

The instructions generally include information as to dosage, dosingschedule, and route of administration for the intended treatment. Thecontainers may be unit doses, bulk packages (e.g., multi-dose packages)or sub-unit doses. Instructions supplied in the kits of the inventionare typically written instructions on a label or package insert (e.g., apaper sheet included in the kit), but machine-readable instructions(e.g., instructions carried on a magnetic or optical storage disk) arealso acceptable.

The label or package insert indicates that the composition is used fortreating, e.g., a neurodegenerative disease. Instructions may beprovided for practicing any of the methods described herein.

The kits of this invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Alsocontemplated are packages for use in combination with a specific device,such as an inhaler, nasal administration device (e.g., an atomizer) oran infusion device such as a minipump. A kit may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Thecontainer may also have a sterile access port (e.g., the container maybe an intravenous solution bag or a vial having a stopper pierceable bya hypodermic injection needle). At least one active agent in thecomposition is an inhibitor of classical complement pathway. Thecontainer may further comprise a second pharmaceutically active agent.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container.

The invention will be more fully understood by reference to thefollowing Examples. They should not, however, be construed as limitingthe scope of the invention. All citations throughout the disclosure arehereby expressly incorporated by reference.

EXAMPLES Example 1: Production of Anti-C1q Antibodies

The anti-C1q antibody M1 was generated by Antibody Solutions Inc.(Sunnyvale Calif.) by immunizing C1q knockout mice with human C1q usingstandard mouse immunization and hybridoma screening technologies(Milstein, C (1999). Bioessays 21: 966-73; Mark Page, Robin Thorpe, TheProtein Protocols Handbook 2002 Editors: John M. Walker, pp 1111-1113).

The anti-C1q antibodies 1C7, 2A1, 3A2, and 5A3 were generated atImmunoPrecise Ltd (Victoria, BC Canada) by immunizing mice with humanC1q protein purified from human plasma (Complement Technology Inc. TylerTexas, Cat #A-099). In brief, female BALB/c mice were injectedintraperitoneal with 25 μg of protein in complete Freund's adjuvant(CFA) on Day 0 and boosts were done with 25 μg of C1q enzyme inincomplete Freund's adjuvant (IFA) on days 21, 42, 52, and a finalintravenous boost on Day 63. Four days following the final boost themice were euthanized, spleens removed and splenocytes were fused withthe myeloma cell line SP2/0. Fused cells were grown onhypoxanthine-aminopterin-thymidine (HAT) selective semisolid media for10-12 days and the resulting hybridomas clones were transferred to96-well tissue culture plates and grown in HAT medium until the antibodytiter was high. The antibody-rich supernatants of the clones wereisolated and tested in an ELISA assay for reactivity with C1q. Positiveclones were isotyped and cultured for 32 days (post HAT selection) toidentify stable expressing clones.

A hybridoma cell line producing the anti-C1q antibody M1 and referred toas mouse hybridoma C1q-M1 7788-1(M) 051613 was deposited with ATCC onJun. 6, 2013 having ATCC Accession Number PTA-120399. M1 was shown tobind specifically to human and mouse C1q and to neutralize biologicalfunctions of C1q, such as complement mediated hemolysis (see, e.g.,Example 3).

Example 2: Anti-C1q Antibodies Specifically Bind to C1q

ELISA Screening

Anti-C1q antibodies 1C7, 2A1, 3A2, and 5A3 were screened for C1q-bindingusing standard ELISA protocols.

Briefly, the day before the assay was performed, 96-well microtiterplates were coated at 0.2 μg/well of C1q-enzyme antigen in 100 μL/wellcarbonate coating buffer pH 9.6 overnight at 4° C. Control wells werecoated with human transferrin. Next, the plates were blocked with 3%milk powder in PBS for 1 hour at room temperature. Then, hybridomatissue culture supernatants were plated at 100 μL/well for 1 hour at 37°C. with shaking. The secondary antibody (1:10,000 goat anti-mouseIgG/IgM(H+L)-HRP) was applied at 100 μL/well for 1.5 hours at roomtemperature with shaking. TMB substrate was added at 50 μL/well for 5minutes at room temperature in the dark. The reaction was stopped with50 μL/well 1M HCl and absorbance readings were taken at a wavelength of450 nm.

Four hybridoma supernatants containing the anti-C1q antibodies 1C7, 2A1,3A2, and 5A3 were tested for binding to human C1q (FIG. 1). By ELISA,all four supernatants showed strong binding signals in the presence ofhuman C1q, whereas only background signals were observed in controlwells containing human transferrin. This experiment demonstrated thatthe anti-C1q antibodies 1C7, 2A1, 3A2, and 5A3 specifically bind tohuman C1q.

Kinetic Analyses

The interactions of the full length anti-C1q antibody M1 with human andmouse C1q proteins were first measured in a kinetic mode andthermodynamic dissociation constants were subsequently calculated.Additionally, M1 binding data was compared with corresponding dataobtained using the reference antibody 4A4B11. 4A4B11 is described inU.S. Pat. No. 4,595,654. The 4A4B11 producing hybridoma cell line isavailable from ATCC (ATCC HB-8327TM).

C1q-antibody interactions were measured using an OCTET™ System accordingto standard protocols and manufacturer's instructions. Briefly, humanand mouse C1q proteins were immobilized separately on a biosensor atthree concentrations (3 nM, 1.0 nM, and 0.33 nM). Next, the anti-C1qantibody M1 was injected onto the C1q-coated biosensor at aconcentration of 2.0 μg/ml and the association constants (k_(on)) anddissociation constants (k_(off)) for anti-C1q antibodies M1 and 4A4B11were measured. The data were fit by non-linear regression analysis andusing the Octet Data Analysis software to yield affinity (K_(D)) andkinetic parameters (k_(on/off)) for the interactions of M1 and 4A4B11with human and mouse C1q respectively (see Table B).

TABLE B Kinetic Analysis of M1 and 4A4B11 Antibody Antigen K_(D) (M)k_(on) (1/Ms) k_(off) (1/s) M1 human C1q 1.28*10⁻¹¹ 5.18*10⁶ 6.31*10⁻⁵M1 mouse C1q 3.23*10⁻¹¹ 1.81*10⁶ 5.84*10⁻⁵ 4A4B11 human C1q 2.29*10⁻¹¹4.49*10⁶ 1.03*10⁻⁴ 4A4B11 mouse C1q undetectable undetectableundetectable

In this experimental series, anti-C1q antibody M1 was shown to bind bothhuman and mouse C1q proteins with very high affinities (K_(D)<10⁻¹⁰ M).By comparison, the reference antibody 4A4B11 was found to bind to humanC1q, whereas binding to mouse C1q was undetectable. Whereas theaffinities of M1 and 4A4B11 for human C1q were on the same order ofmagnitude (i.e., in the double-digit picomolar range; K_(D)˜10-30 pM),the affinity of M1 for mouse C1q was determined to be about four ordersof magnitude higher (K_(D)˜30 pM) than that the affinity of 4A4B11 formouse C1q (K_(D)˜40 nM).

Anti-C1q Antibodies M1 and 4A4B11 do not Compete for C1q-Binding

Blocking experiments were performed to determine whether the anti-C1qantibodies M1 and 4A4B11 bind to the same or overlapping epitopes ofhuman C1q or whether M1 and 4A4B11 bind to separate C1q epitopes.

To this end, M1 was coated on a biosensor chip (BIACORE™) andsubsequently contacted with a combination of human C1q and M1, acombination of human C1q and 4A4B11, or human C1q alone. C1q-binding toM1 was followed for 10 min and dissociation of M1-C1q complexes wassubsequently followed for 20 min. Relative binding signals were recordedat the end of the association and dissociation periods. Table C showsthe results of these experiments.

TABLE C Analysis of Simultaneous Interactions of M1 and 4A4B11 withhuman C1q Sensor Association Ab Antigen Solution ResponseDissociationResponse ID: ID: Ab ID: (nm) @ 600 s (nm) @ 1200 s M1 hC1qM1 −0.0119 −0.00945 M1 hC1q 4A4B11 0.8213 0.82139 M1 hC1q None 0.47150.45137 (Ag Only)

It was found that C1q alone bound effectively to immobilized M1 antibodyon the biosensor chip. Preincubation of C1q with soluble M1 antibodyprevented all binding of the resulting M1-C1q complex to immobilized M1.By contrast, preincubation of C1q with 4A4B11 did not prevent theinteraction of the resulting 4A4B11-C1q complex with immobilized M1. Thelarger relative binding signals observed in the binding experimentinvolving the 4A4B11-C1q complex relative to the binding experimentinvolving C1q alone is due to the fact that the relative binding signalscorrelate with the molecular weight of the soluble binding partners andthat the 4A4B11-C1q complex has a higher molecular weight than C1qalone.

These results demonstrate that 4A4B11 does not compete with M1 for C1qbinding. Therefore, 4A4B11 and M1 may recognize separate epitopes onC1q.

Example 3: Anti-C1q Antibodies Inhibit Complement-Mediated Hemolysis

Anti-C1q antibodies were tested in human and rodent hemolytic assays(CH50) for their ability to neutralize C1q and block its activation ofthe downstream complement cascade. CH50 assays were conductedessentially as described in Current Protocols in Immunology (1994)Supplement 9 Unit 13.1. In brief, 5 microliters (μ1) of human serum(Cedarlane, Burlington, N.C.), 0.625 μl of Wistar rat serum, or 2.5 μlof C57Bl/6 mouse serum was diluted to 50 μl of GVB buffer (Cedarlane,Burlington, N.C.) and added to 50 μl of the monoclonal antibodies (1 μg)diluted in GVB buffer. The antibody:serum mixture was pre-incubated for30 minutes on ice and then added to 100 μl of EA cells (2×10⁸/ml) forrat and human assays, and 4×10⁷/ml for mouse assays. The EA cells weregenerated exactly as specified in Current Protocols using Sheeps bloodin Alsever's (Cedarlane Cat #CL2581) and hemolysin (Cedalane Cat#CL9000). The EA cells, serum and antibody mixture was incubated for 30minutes at 37° C. and then placed on ice. Next 1.2 ml of 0.15 M NaCl wasadded to the mixture and the OD₄₁₂ of the sample was read in aspectrophotometer to determine the amount of cell lysis. The percentinhibition of the test antibodies was determined relative to a controlmouse IgG1 antibody (Abcam ab18447).

A modified CH50 assay (also referred to as C1F hemolysis assay) wasperformed that provided limiting quantities of the C1 complex from humanserum to provide greater sensitivity for assessing C1 activity andpotential C1 inhibition. In brief, the assay was conducted as follows.First, 3×10⁷ sheep red blood cells (RBC) were incubated with anti-sheepRBC IgM antibody to generate activated erythrocytes (EA cells). The EAcells were then incubated with purified C4b protein to create EAC4bcells. EAC4b cells were subsequently incubated with diluted(1:1000-1:10000) normal human serum (NHS) that was pre-incubated with orwithout anti-C1q and control mouse IgG antibodies, to provide a limitingquantity of human C1. Next, the resulting EAC14 cells were incubatedwith purified human C2 protein to generate EAC14b2a cells. Finally,guinea pig serum was added in an EDTA buffer and incubated at 37° C. for30 minutes. Cell lysis was measured in a spectrophotometer at awavelength of 450 nm.

First, four C1q-binding antibodies (1C7, 2A1, 3A2, and 5A3) were testedin the human CH50 assay at a single concentration (1 μg) (FIG. 2). Allfour antibodies were found to inhibit hemolysis. The anti-C1q antibody1C7 inhibited hemolysis at greater than 90%, 2A1 inhibited hemolysis atgreater than 40%, 3A2 inhibited hemolysis at greater than 60%, and 5A3inhibited hemolysis at greater than 50%.

Next, anti-C1q antibodies 1C7 and 3A2 were tested in the human CH50hemolysis assay in a dose-response format (FIG. 3). Anti-C1q antibody4A4B11 was used as a reference. Both 1C7 and 3A2 antibodies inhibitedCH50 hemolysis in a dose-dependent manner. Approximately 100 ng of the1C7 antibody and approximately 200 ng of the 3A2 were required toinhibit 50% of the hemolysis observed (FIG. 3).

Anti-C1q antibody M1 was tested for its C1q neutralizing activity inhuman, mouse, and rat CH50 assays (FIG. 4A-C). Testing was conducted indose-response formats. Anti-C1q antibody 4A4B11 was used as a reference.M1 was demonstrated to neutralize C1q activity in human, mouse, and ratCH50 hemolysis assays in a dose-dependent manner (FIG. 4A-4C). Bycontrast, 4A4B11 was found to neutralize C1q activity only in the humanCH50 assay, whereas the reference antibody was inactive in the mouse andrat CH50 hemolysis assays (up to 2 μg). In the human and rat CH50hemolysis assays M1 inhibited greater than 90% and up to 100% ofhemolysis (FIGS. 4A and 4C); in the mouse assay M1 inhibited greaterthan 50% of hemolysis (FIG. 4B). In the human CH50 assay, less than 125ng of M1 were required to achieve 50% inhibition of hemolysis. In themouse CH50 assay, approximately 500 ng of M1 were required to achieve50% inhibition of hemolysis. In the rat CH50 assay, less than 16 ng wererequired to achieve 50% inhibition of hemolysis.

Example 4: Epitope Mapping for Antibody 4A4B11 and M1

In order to determine the nature of the epitope (i.e., linear orconformational), the inhibition of the interaction between the C1Qprotein and the antibodies 4A4B11 (ANN-001) and M1 (ANN-005) byunstructured peptides generated by proteolysis of the C1q antigen wasevaluated. If the peptides generated by complete proteolysis of theantigen are able to inhibit the binding of the antigen on the antibody,the interaction is not based on conformation, and the epitope is linear.If the peptides generated by complete proteolysis of the antigen areunable to inhibit the binding of the antigen on the antibodies 4A4B11and M1, the conformation is necessary for interaction. Based on the datadescribed in detail below, unstructured peptides generated by digestionof native C1q did not compete with intact C1q for binding to the 4A4B11(ANN-001) and M1 (ANN-005) antibodies (see FIG. 2), suggesting that theC1q epitope for these antibodies is a complex conformational epitope.

In order to determine the key residues of the conformational C1q epitopethat binds of ANN-001 and ANN-005 on C1Q antigen with high resolutionthe antibody/antigen complexes were incubated with deuteratedcross-linkers and subjected to multi-enzymatic proteolytic cleavage.After enrichment of the cross-linked peptides, the samples were analyzedby high resolution mass spectrometry (nLC-Orbitrap MS) and the datagenerated analyzed using XQuest software. The analysis described belowindicates that antibibody 4A4B11 (ANN-001) binds to an epitope thatincludes amino acids 5202 and K219 of human C1QA and Y225 of human C1QC,and antibody M1 (ANN-005) binds to an epitope that includes amino acidK219 of human C1QA and S185 of human C1QC. See the amino acid sequencealignment of human and mouse C1qA and C1qC as shown below.

Amino Acid Sequence Alignment of Human and Mouse C1qA

MEGPRGWLVLCVLAISLASMVTEDLCRAPDGKKGEAGRPGRRGRPGLKGEQGEPGAPGIR humanMETSQGWLVACVLTMTLVWTVAEDVCRAPNGKDGAPGNPGRPGRPGLKGERGEPGAAGIR mouse** .:**** ***:::*.  *:**:****:**.* .*.*** ********:*****.***TGIQGLKGDQGEPGPSGNPGKVGYPGPSGPLGARGIPGIKGTKGSPGNIKDQPRPAFSAI humanTGIRGFKGDPGESGPPGKPGNVGLPGPSGPLGDSGPQGLKGVKGNPGNIRDQPRPAFSAI mouse***:*:*** **.**.*:**:** ********  *  *:**.**.****:**********RRNPPMGGNVVIFDTVITNQEEPYQNHSGRFVCTVPGYYYFTFQVLSQWEICLSIVSSSR humanRQNPMTLGNVVIFDKVLTNQESPYQNHTGRFICAVPGFYYFNFQVISKWDLCLFIKSSSG mouse*:**   *******.*:****.*****:***:*:***:***.***:*:*::** * ***GQVRRSLGFCDTTNKGLFQVVSGGMVLQLQQGDQVWVEKDPKKGHIYQGSEADSVFSGFL humanGQPRDSLSFSNTNNKGLFQVLAGGTVLQLRRGDEVWIEKDPAKGRIYQGTEADSIFSGFL mouse** * **.*.:*.*******::** ****::**:**:**** **:****:****:*****IFPSA human (SEQ ID NO: 1) IFPSA mouse (SEQ ID NO: 14) *****Amino Acid Sequence Alignment of Human and Mouse C1qC

MDVGPSSLPHLGLKLLLLLLLLP-LRGQANTGCYGIPGMPGLPGAPGKDGYDGLPGPKGE humanMVVGPSCQPPCGLCLLLLFLLALPLRSQASAGCYGIPGMPGMPGAPGKDGHDGLQGPKGE mouse* ****. ** ** ****:**   **.**.:**********:********:*** *****PGIPAIPGIRGPKGQKGEPGLPGHPGKNGPMGPPGMPGVPGPMGIPGEPGEEGRYKQKFQ humanPGIPAVPGTRGPKGQKGEPGMPGHRGKNGPRGTSGLPGDPGPRGPPGEPGVEGRYKQKHQ mouse*****:** ***********:*** ***** *  *:** *** * ***** ******* *SVFTVTRQTHQPPAPNSLIRFNAVLTNPQGDYDTSTGKFTCKVPGLYYFVYHASHTANLC humanSVFTVTRQTTQYPEANALVRFNSVVTNPQGHYNPSTGKFTCEVPGLYYFVYYTSHTANLC mouse********* * * .*:*:***:*:*****.*:.*******:*********::*******VLLYRSGVKVVTFCGHTSKTNQVNSGGVLLRLQVGEEVWLAVNDYYDMVGIQGSDSVFSG humanVHLNLNLARVASFCDHMFNSKQVSSGGVLLRLQRGDEVWLSVNDYNGMVGIEGSNSVFSG mouse* *  . .:*.:**.*  :::**.********* *;****;**** .****:**:*****FLLFPD human (SEQ ID NO: 3) FLLFPD mouse (SEQ ID NO: 15) ******1. Identification of the C1q/Antibody Complexes by Mass Spectrometry

The C1q/antibody complexes were generated by mixing equimolar solutionsof C1q antigen and antibody (4 μM in 5 μl each). One μl of the mixtureobtained was mixed with 1 μl of a matrix composed of a re-crystallizedsinapinic acid matrix (10 mg/ml) in acetonitrile/water (1:1, v/v), TFA0.1% (K200 MALDI Kit). After mixing, 1 μl of each sample was spotted onthe MALDI plate (SCOUT 384). After crystallization at room temperature,the plate was introduced in the MALDI mass spectrometer and analysedimmediately. The analysis has been repeated in triplicate. FIG. 5A andFIG. 5B show the presence of the antigen, antibody and antigen/antibodycomplexes for C1q/4A4B11 (FIG. 5A) and C1q/M1 (FIG. 5B). Peaks arepresent at the predicted molecular weights of monomeric antibody (˜150kDa) and C1q monomer (˜460 kDa) and there is a 1:1 complex ofantibody:antigen present at ˜615 kDa.

2. Unstructured C1q Peptides Generated by Proteolysis do not Compete forBinding of C1q to Antibody

To determine if the C1q/antibody complexes could be competed withpeptides the C1q antigen was digested with immobilized pepsin. 25 μl ofthe antigen with a concentration of 10 μM were mixed with immobilizedpepsin 5 μM and incubate at room temperature for 30 minutes. After theincubation time the sample was centrifuged and the supernatant waspipetted. The completion of the proteolysis was controlled by High-MassMALDI mass spectrometry in linear mode. The pepsin proteolysis wasoptimized in order to obtain a large amount of peptide in the 1000-3500Da range. Next, 5 μl of the antigen peptides generated by proteolysiswere mixed with 5 μl of ANN-001 or ANN-005 (8 μM) and incubated at 37°C. for 6 hours. After incubation of ANN-001 or ANN-005 with the C1Qantigen peptides, 5 μl of the mixture was mixed with 5 μl of the C1Qantigen (4 μM) so the final mix contained 2 μM/2 μM/2.5 μM of C1Qantigen/4A4B11 or M1/C1Q antigen Peptides.

The MALDI ToF MS analysis was performed using CovalX's HM3 interactionmodule with a standard nitrogen laser and focusing on different massranges from 0 to 2000 kDa. For the analysis, the following parametershave been applied for Mass Spectrometer: Linear and Positive mode; IonSource 1: 20 kV; Ion Source 2: 17 kV; Pulse Ion Extraction: 400 ns; forHM3: Gain Voltage: 3.14 kV; Gain Voltage: 3.14 kV; Acceleration Voltage:20 kV.

To calibrate the instrument, an external calibration with clusters ofInsulin, BSA and IgG has been applied. For each sample, 3 spots wereanalyzed (300 laser shots per spots). The presented spectrum correspondsto the sum of 300 laser shots. The MS data were analyzed using theComplex Tracker analysis software version 2.0 (CovalX Inc).

The results are shown in FIG. 6A and FIG. 6B, and demonstrate that C1qpeptides do not compete with intact C1q for binding to monoclonalantibody ANN-005 (M1).

3. Identification of the Conformational Epitopes for C1q Binding toANN-001 and ANN-005

Using chemical cross-linking, High-Mass MALDI mass spectrometry andnLC-Orbitrap mass spectrometry the interaction interface between theantigen C1Q and two monoclonal antibodies ANN-001 and ANN-005 wascharacterized. 5 μl of the sample C1Q antigen (concentration 4 μM) wasmixed with 5 μl of the sample ANN-001 (Concentration 4 μM) or ANN-005(Concentration 4 μM) in order to obtain an antibody/antigen mix withfinal concentration 2 μM/2 μM. The mixture was incubated at 37° C. for180 minutes. In a first step, 1 mg of DiSuccinimidylSuberate H12(DSS-H12) cross-linker was mixed with 1 mg of DiSuccinimidylSuberate D12(DSS-D12) cross-linker. The 2 mg prepared were mixed with 1 ml of DMF inorder to obtain a 2 mg/ml solution of DSS H12/D12. 10 μl of theantibody/antigen mix prepared previously were mixed with 1 μl of thesolution of cross-linker d0/d12 prepared (2 mg/ml). The solution wasincubated 180 minutes at room temperature in order to achieve thecross-linking reaction. In order to facilitate the proteolysis, it wasnecessary to reduce the disulfide bound present in this protein. Thecross-linked sample was mixed with 20 μl of ammonium bicarbonate (25 mM,pH 8.3). After mixing 2.5 μl of DTT (500 mM) is added to the solution.The mixture was then incubated 1 hour at 55° C. After incubation, 2.5 μlof iodioacetamide (1 M) was added before 1 hour of incubation at roomtemperature in a dark room. After incubation, the solution was diluted1/5 by adding 120 μl of the buffer used for the proteolysis. 145 μl ofthe reduced/alkyled cross-linked sample was mixed with 2 μl of trypsin(Sigma, T6567). The proteolytic mixture was incubated overnight at 37°C. For a-chymotrypsin proteolysis, the buffer of proteolysis wasTris-HCL 100 mM, CaCl₂ 10 mM, pH7.8. The 145 μl of the reduced/alkyledcross-linked complex was mixed with 2 μl of α-chymotrypsin 200 μM andincubated overnight at 30° C. For this analysis, an nLC in combinationwith Orbitrap mass spectrometry were used. The cross-linker peptideswere analyzed using Xquest version 2.0 and stavrox software. Thepeptides identified and cross-linked amino acids are indicated in TableD below.

TABLE DC1q cross-linked peptides and contact residues necessary for ANN-001 andANN-005 binding Protease C1q Contact Digest X-linked Peptide SubunitResidue Antibody Trypsin GLFQVVSGGMVLQLQQGDQVWVEK C1qA K219 ANN-001(SEQ ID NO: 25, residues 196-219 of SEQ ID NO: 1) TrypsinFQVVSGGMVLQL (SEQ ID NO: 26, C1qA S202 ANN-001residues 198-209 of SEQ ID NO: 1) Chymotrypsin YDMVGIQGSDSVFSGF C1qCY225 ANN-001 (SEQ ID NO: 21, residues 225-240 of SEQ ID NO: 3) TrypsinGLFQVVSGGMVLQLQQGDQVWVEK C1qA K219 ANN-005(SEQ ID NO: 25, residues 196-219 of SEQ ID NO: 1) ChymotrypsinRSGVKVVTF (SEQ ID NO: 24, residues C1qC S185 ANN-005184-192 of SEQ ID NO: 3)4. Light Chain and Heavy Chain Variable Domain Sequences of Antibody M1

Using standard techniques, the nucleic acid and amino acid sequencesencoding the light chain variable and the heavy chain variable domain ofantibody M1 were determined. T

he amino acid sequence of the light chain variable domain of antibody M1is: DVQITQSPSYLAASPGETITINCRASKSINKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPLTFGAGTKLELK (SEQ ID NO:4). The hypervariable regions (HVRs) of the light chain variable domain are depictedin bolded and underlined text. In some embodiments, the HVR-L1 of the M1light chain variable domain has the sequence RASKSINKYLA (SEQ ID NO:5),the HVR-L2 of the M1 light chain variable domain has the sequenceSGSTLQS (SEQ ID NO:6), and the HVR-L3 of the M1 light chain variabledomain has the sequence QQHNEYPLT (SEQ ID NO:7).

The amino acid sequence of the heavy chain variable domain of antibodyM1 is: QVQLQQPGAELVKPGASVKLSCKSSGYHFTSYWMHWVKQRPGQGLEWIGVIHPNSGSINYNEKFESKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAGERDSTEVLPMDYWGQG TSVTVSS (SEQID NO:8). The hyper variable regions (HVRs) of the heavy chain variabledomain are depicted in bolded and underlined text. In some embodiments,the HVR-H1 of the M1 heavy chain variable domain has the sequenceGYHFTSYWMH (SEQ ID NO:9), the HVR-H2 of the M1 heavy chain variabledomain has the sequence VIHPNSGSINYNEKFES (SEQ ID NO:10), and the HVR-H3of the M1 heavy chain variable domain has the sequence ERDSTEVLPMDY (SEQID NO:11).

The nucleic acid sequence encoding the light chain variable domain wasdetermined to be:

(SEQ ID NO: 12) GATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAGAAACCATTACTATTAATTGCAGGGCAAGTAAGAGCATTAACAAATATTTAGCCTGGTATCAAGAGAAACCTGGGAAAACTAATAAGCTTCTTATCTACTCTGGATCCACTTTGCAATCTGGAATTCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACTCTCACCATCAGTAGCCTGGAGCCTGAAGATTTTGCAATGTATTACTGTCAACAACATAATGAATACCCGCTCACGTTCGGTGCTGGGACC AAGCTGGAGCTGAAA.The nucleic acid sequence encoding the heavy chain variable domain wasdetermined to be:

(SEQ ID NO: 13) CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGTCTTCTGGCTACCATTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGTGATTCATCCTAATAGTGGTAGTATTAACTACAATGAGAAGTTCGAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCGGCGGTCTATTATTGTGCAGGAGAGAGAGATTCTACGGAGGTTCTCCCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTC TCCTCA.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

Deposit of Material

The following materials have been deposited according to the BudapestTreaty in the American Type Culture Collection, ATCC Patent Depository,10801 University Blvd., Manassas, Va. 20110-2209, USA (ATCC):

Deposit ATCC Sample ID Isotype Date Accession No. Mouse hybridoma IgG1,Jun. 6, 2013 PTA-120399 C1qM1 7788-1(M) kappa 051613 producing anti-C1qantibody M1

The hybridoma cell line producing the M1 antibody (mouse hybridoma C1qM17788-1(M) 051613) has been deposited with ATCC under conditions thatassure that access to the culture will be available during pendency ofthe patent application and for a period of 30 years, or 5 years afterthe most recent request, or for the effective life of the patent,whichever is longer. A deposit will be replaced if the deposit becomesnonviable during that period. The deposit is available as required byforeign patent laws in countries wherein counterparts of the subjectapplication, or its progeny are filed. However, it should be understoodthat the availability of the deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

What is claimed is:
 1. A method of treating a disease associated withC1q mediated activation of the classical complement pathway, the methodcomprising administering a therapeutically effective dose of an anti-C1qantibody, comprising (a) a light chain variable domain and a heavy chainvariable domain, wherein the light chain variable domain comprises thehypervariable region (HVR)-L1, HVR-L2, and HVR-L3 of the monoclonalantibody M1 produced by a hybridoma cell line with ATCC Accession NumberPTA-120399; and wherein the heavy chain variable domain comprises theHVR-H1, HVR-H2, and HVR-H3 of the monoclonal antibody M1 produced by ahybridoma cell line with ATCC Accession Number PTA-120399; or (b) aheavy chain HVR selected from HVR-H1 comprising the amino acid sequenceof SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO:10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:
 11. 2.The method of claim 1, wherein the disease associated with C1q mediatedactivation of the classical complement pathway is a neurodegenerativedisorder.
 3. The method of claim 2, wherein the neurodegenerativedisorder is associated with loss of synapses or loss nerve connections.4. The method of claim 2, wherein the neurodegenerative disorder isassociated with synapse loss that is dependent on complement receptor 3(CR3) or complement receptor CR1.
 5. The method of claim 2, wherein theneurodegenerative disorder is associated with pathologicalactivity-dependent synaptic pruning.
 6. The method of claim 2, whereinthe neurodegenerative disorder is associated with synapse phagocytosisby microglia.
 7. The method of claim 2, wherein the neurodegenerativedisorder is Alzheimer's disease, amyotrophic lateral sclerosis, multiplesclerosis, glaucoma, myotonic dystrophy, Down syndrome, Parkinson'sdisease, or Huntington's disease.
 8. The method of claim 1, wherein thedisease associated with C1q mediated activation of the classicalcomplement pathway is an inflammatory disease, autoimmune disease, ormetabolic disorder.
 9. The method of claim 8, wherein the inflammatorydisease, autoimmune disease, or metabolic disorder is diabetes, obesity,rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS),remote tissue injury after ischemia and reperfusion, complementactivation during cardiopulmonary bypass surgery, dermatomyositis,pemphigus, lupus nephritis and resultant glomerulonephritis andvasculitis, cardiopulmonary bypass, cardioplegia-induced coronaryendothelial dysfunction, type II membranoproliferativeglomerulonephritis, IgA nephropathy, acute renal failure,cryoglobulemia, antiphospholipid syndrome, macular degenerativediseases, age-related macular degeneration (AMD), choroidalneovascularization (CNV), uveitis, diabetic retinopathy,ischemia-related retinopathy, endophthalmitis, intraocular neovasculardisease, diabetic macular edema, pathological myopia, von Hippel-Lindaudisease, histoplasmosis of the eye, Neuromyelitis Optica (NMO), CentralRetinal Vein Occlusion (CRVO), corneal neovascularization, retinalneovascularization, allo-transplantation, hyperacute rejection,hemodialysis, chronic occlusive pulmonary distress syndrome (COPD),asthma, or aspiration pneumonia.
 10. The method of claim 1, wherein thedisease associated with C1q mediated activation of the classicalcomplement pathway is myasthenia gravis, Diabetes mellitus type 1,Hashimoto's thyroiditis, Addison's disease, Coeliac disease, Crohn'sdisease, pernicious anaemia, Pemphigus vulgaris, vitiligo, autoimmunehemolytic anemias, paraneoplastic syndromes, a vasculitis disease,polymyalgia rheumatica, temporal arteritis, or Wegener's granulomatosis.11. The method of claim 1, wherein the antibody comprises a light chainvariable domain comprising the amino acid sequence of SEQ ID NO:4. 12.The method of claim 1, wherein the antibody comprises a heavy chainvariable domain comprising the amino acid sequence of SEQ ID NO:8. 13.The method of claim 1, wherein the antibody binds specifically to humanC1q, mouse C1q, and rat C1q.
 14. The method of claim 1, wherein theantibody has a dissociation constant (K_(D)) for human C1q and mouse C1qless than about 30 nM.
 15. The method of claim 1, wherein the antibodyspecifically binds to C1q and inhibits CH50 hemolysis.
 16. The method ofclaim 15, wherein CH50 hemolysis comprises human, mouse, or rat CH50hemolysis.
 17. The method of claim 15, wherein the antibody is capableof neutralizing at least 50%, at least 80%, or at least 90% of CH50hemolysis.
 18. The method of claim 15, wherein the antibody is capableof neutralizing at least 50% of CH50 hemolysis at a dose of less than200 ng/ml, less than 100 ng/ml, less than 50 ng/ml, or less than 20ng/ml.
 19. The method of claim 1, wherein the antibody is a murine,humanized, or chimeric antibody.
 20. The method of claim 11, wherein theantibody comprises a heavy chain variable domain comprising the aminoacid sequence of SEQ ID NO:8.
 21. The method of claim 11, wherein theantibody has a dissociation constant (K_(D)) for human C1q and mouse C1qless than about 30 nM.
 22. The method of claim 12, wherein the antibodyhas a dissociation constant (K_(D)) for human C1q and mouse C1q lessthan about 30 nM.
 23. The method of claim 11, wherein the antibody is anantibody fragment, wherein the antibody fragment is a Fab, F(ab′)₂ orFab′ fragment.
 24. The method of claim 12, wherein the antibody is anantibody fragment, wherein the antibody fragment is a Fab, F(ab′)₂ orFab′ fragment.
 25. The method of claim 20, wherein the antibody is anantibody fragment, wherein the antibody fragment is a Fab, F(ab′)₂ orFab′ fragment.
 26. The method of claim 1, wherein the antibody is of theIgG class.
 27. The method of claim 26, wherein the antibody has an IgG1,IgG2, IgG3, or IgG4 isotype.
 28. The method of claim 1, wherein theantibody specifically binds to and neutralizes a biological activity ofC1q.
 29. The method of claim 28, wherein the biological activity is (1)C1q binding to an autoantibody, (2) C1q binding to C1r, (3) C1q bindingto C1s, (4) C1q binding to phosphatidylserine, (5) C1q binding topentraxin-3, (6) C1q binding to C-reactive protein (CRP), (7) C1qbinding to globular C1q receptor (gC1qR), (8) C1q binding to complementreceptor 1 (CR1), (9) C1q binding to beta-amyloid, or (10) C1q bindingto calreticulin.
 30. The method of claim 28, wherein the biologicalactivity is (1) activation of the classical complement activationpathway, (2) activation of antibody and complement dependentcytotoxicity, (3) CH50 hemolysis, (4) synapse loss, (5) B-cell antibodyproduction, (6) dendritic cell maturation, (7) T-cell proliferation, (8)cytokine production (9) microglia activation, (10) Arthus reaction, (11)phagocytosis of synapses or nerve endings, or (12) activation ofcomplement receptor 3 (CR3/C3) expressing cells.